Methods for diagnosis, prognosis and methods of treatment

ABSTRACT

This invention is directed to methods and compositions for diagnosis, prognosis and for determining methods of treatment. The physiological status of cells present in a sample (e.g. clinical sample) can be used in diagnosis or prognosis of a condition (e.g. Chronic Lymphocytic Leukemia), in patient selection for therapy, to monitor treatment and to modify or optimize therapeutic regimens. The physiological status of a cell can be determined by comparing the intracellular status of one or more activation elements (e.g. the phosphorylation status of a signaling molecule) in a cell (e.g. a cancer cell) to that of another cell (e.g. a normal cell). The physiological status of a cell can be further classified by adding one or more modulators (e.g. an inhibitor or activator) to the cell in question. In some embodiments, the invention is directed to methods of determining a phenotypic profile of a population of cells.

CROSS-REFERENCE

This application is a Continuation Application which claims the benefitof U.S. application Ser. No. 12/229,476 filed Aug. 21, 2008; whichclaims the benefit of U.S. Provisional Application No. 60/957,160 filedAug. 21, 2007 and U.S. Provisional Application No. 61/048,920 filed Apr.29, 2008 and each of which is hereby expressly incorporated by referencein its entirety.

BACKGROUND OF THE INVENTION

Many conditions are characterized by disruptions in cellular pathwaysthat lead, for example, to aberrant control of cellular processes, or touncontrolled growth and proliferation of cells. These disruptions areoften caused by changes in the activity of molecules participating incellular pathways. For example, specific signaling pathway alterationshave been described for many cancers. Despite the increasing evidencethat disruption in cellular pathways mediate the detrimentaltransformation, the precise molecular events underlying thesetransformations have not been elucidated. As a result, therapeutics maynot be effective in treating conditions involving cellular pathways thatare not well understood. Thus, the successful diagnosis of a conditionand use of therapies will require knowledge of the cellular events thatare responsible for the condition pathology.

In addition, patients suffering from different conditions followheterogeneous clinical courses. For instance, tremendous clinicalvariability among remissions is also observed in cancer patients, eventhose that occur after one course of therapy. Some leukemia patientssurvive for prolonged periods without definitive therapy, while othersdie rapidly despite aggressive treatment. Patients who are resistant totherapy have very short survival times, regardless of when theresistance occurs. While various staging systems have been developed toaddress this clinical heterogeneity, they cannot accurately predictwhether an early or intermediate stage patient will experience anindolent or aggressive course of disease.

Accordingly, there is a need for a reliable indicator of an individualpredicted disease course to help clinicians to identify those patientsthat will respond to treatment, patients that progress to a moreadvanced state of the disease and patients with emerging resistance totreatment.

SUMMARY OF THE INVENTION

Other objects, features and advantages of the methods and compositionsdescribed herein will become apparent from the following detaileddescription. It should be understood, however, that the detaileddescription and the specific examples, while indicating specificembodiments, are given by way of illustration only, since variouschanges and modifications within the spirit and scope of the inventionwill become apparent to those skilled in the art from this detaileddescription.

All publications, patents, and patent applications mentioned in thisspecification are herein incorporated by reference to the same extent asif each individual publication or patent application was specificallyand individually indicated to be incorporated by reference.

As disclosed herein is a method for classifying a cell comprisingcontacting the cell with an inhibitor, determining the presence orabsence of a change in activation level of an activatable element in thecell, and classifying the cell based on the presence or absence of thechange in the activation level of the activatable element. In someembodiments the change in activation level of an activatable element isan increase in the activation level of an activatable element. In someembodiments the activatable element is a protein subject tophosphorylation or dephosphorylation.

In some embodiments of the methods, the invention provides a method forclassifying a cell by contacting the cell with an inhibitor; determiningthe activation levels of a plurality of activatable elements in thecell; and classifying the cell based on the activation level. In someembodiments, the inhibitor is a kinase or phosphatase inhibitor, such asadaphostin, AG 490, AG 825, AG 957, AG 1024, aloisine, aloisine A,alsterpaullone, aminogenistein, API-2, apigenin, arctigenin, AY-22989,BAY 61-3606, bisindolylmaleimide IX, chelerythrine,10[4′-(N,N-Diethylamino)butyl]-2-chlorophenoxazine hydrochloride,dasatinib, 2-Dimethylamino-4,5,6,7-tetrabromo-1H-benzimidazole,5,7-Dimethoxy-3-(4-pyridinyl)quinoline dihydrochloride, edelfosine,ellagic acid, enzastaurin, ER 27319 maleate, erlotinib, ET18OCH3,fasudil, flavopiridol, gefitinib, GW 5074, H-7, H-8, H-89, HA-100,HA-1004, HA-1077, HA-1100, hydroxyfasudil, indirubin-3′-oxime,5-Iodotubercidin, kenpaullone, KN-62, KY12420, LFM-A13, lavendustin A,luteolin, LY-294002, LY294002, mallotoxin, ML-9, NSC-154020, NSC-226080,NSC-231634, NSC-664704, NSC-680410, NU6102, olomoucine, oxindole I,PD-153035, PD-98059, PD-169316, phloretin, phloridzin, piceatannol,picropodophyllin, PK1, PP1, PP2, purvalanol A, quercetin, R406, R788,rapamune, rapamycin, Ro 31-8220, roscovitine, rottlerin, SB202190,SB203580, sirolimus, sorafenib, SL327, SP600125, staurosporine, STI-571,SU-11274, SU1498, SU4312, SU6656, 4,5,6,7-Tetrabromotriazole, TG101348,Triciribine, Tyrphostin AG 490, Tyrphostin AG 825, Tyrphostin AG 957,Tyrphostin AG 1024, Tyrphostin SU1498, U0126, VX-509, VX-667, VX-680,W-7, wortmannin, XL-019, XL-147, XL-184, XL-228, XL-281, XL-518, XL-647,XL-765, XL-820, XL-844, XL-880, Y-27632, ZD-1839, ZM-252868, ZM-447439,H₂O₂, siRNA, miRNA, Cantharidin, (−)-p-Bromotetramisole, Microcystin LR,Sodium Orthovanadate, Sodium Pervanadate, Vanadyl sulfate, Sodiumoxodiperoxo(1,10-phenanthroline)vanadate, bis(maltolato)oxovanadium(IV),Sodium Molybdate, Sodium Perm olybdate, Sodium Tartrate, Imidazole,Sodium Fluoride, f3-Glycerophosphate, Sodium Pyrophosphate Decahydrate,Calyculin A, Discodermia calyx, bpV(phen), mpV(pic), DMHV, Cypermethrin,Dephostatin, Okadaic Acid, NIPP-1,N-(9,10-Dioxo-9,10-dihydro-phenanthren-2-yl)-2,2-dimethyl-propionamide,α-Bromo-4-hydroxyacetophenone, 4-Hydroxyphenacyl Br,α-Bromo-4-methoxyacetophenone, 4-Methoxyphenacyl Br,α-Bromo-4-(carboxymethoxy)acetophenone, 4-(Carboxymethoxy)phenacyl Br,and bis(4-Trifluoromethylsulfonamidophenyl)-1,4-diisopropylbenzene,phenyarsine oxide, Pyrrolidine Dithiocarbamate, or Aluminum fluoride. Insome embodiments the phosphatase inhibitor is H₂O₂.

In some embodiments the cell is a hematopoietic-derived cell. In someembodiments, the hematopoietically derived cell is selected from thegroup consisting of pluripotent hematopoietic stem cells, B-lymphocytelineage progenitor or derived cells, T-lymphocyte lineage progenitor orderived cells, NK cell lineage progenitor or derived cells, granulocytelineage progenitor or derived cells, monocyte lineage progenitor orderived cells, megakaryocyte lineage progenitor or derived cells anderythroid lineage progenitor or derived cells. In some embodiments, thehematopoietic derived cell is a B-lymphocyte lineage progenitor andderived cell, e.g., an early pro-B cell, late pro-B cell, large pre-Bcell, small pre-B cell, immature B cell, mature B cell, plasma cell andmemory B cell, a CD5+ B cell, a CD38+ B cell, a B cell bearing amutatated or non mutated heavy chain of the B cell receptor, or a B cellexpressing Zap70.

In some embodiments, the classification includes classifying the cell asa cell that is correlated with a clinical outcome. In some embodiments,the clinical outcome is the prognosis and/or diagnosis of a condition.In some embodiments, the clinical outcome is the presence or absence ofa neoplastic or a hematopoietic condition, such as Non-Hodgkin Lymphoma,Hodgkin or other lymphomas, acute or chronic leukemias, polycythemias,thrombocythemias, multiple myeloma or plasma cell disorders, e.g.,amyloidosis and Waldenstrom's macroglobulinemia, myelodysplasticdisorders, myeloproliferative disorders, myelofibrosis, or atypicalimmune lymphoproliferations. In some embodiments, the neoplastic orhematopoietic condition is non-B lineage derived, such as acute myeloidleukemia (AML), Chronic Myeloid Leukemia (CML), non-B cell acutelymphocytic leukemia (ALL), non-B cell lymphomas, myelodysplasticdisorders, myeloproliferative disorders, myelofibrosis, polycythemias,thrombocythemias, or non-B atypical immune lymphoproliferations. In someembodiments, the neoplastic or hematopoietic condition is a B-Cell or Bcell lineage derived disorder, such as Chronic Lymphocytic Leukemia(CLL), B lymphocyte lineage leukemia, B lymphocyte lineage lymphoma,Multiple Myeloma, acute lymphoblastic leukemia (ALL), B-cellpro-lymphocytic leukemia, precursor B lymphoblastic leukemia, hairy cellleukemia or plasma cell disorders, e.g., amyloidosis or Waldenstrom'smacroglobulinemia, B cell lymphomas including but not limited to diffuselarge B cell lymphoma, follicular lymphoma, mucosa associated lymphatictissue lymphoma, small cell lymphocytic lymphoma and mantle celllymphoma. In some embodiments, the condition is CLL. In someembodiments, the CLL is defined by a monoclonal B cell population thatco-expresses CD5 with CD19 and CD23 or CD5 with CD20 and CD23 and bysurface immunoglobulin expression.

In some embodiments, the clinical outcome is the staging or grading of aneoplastic or hematopoietic condition. Examples of staging in methodsprovided by the invention include aggressive, indolent, benign,refractory, Roman Numeral staging, TNM Staging, Rai staging, Binetstaging, WHO classification, FAB classification, IPSS score, WPSS score,limited stage, extensive stage, staging according to cellular markerssuch as ZAP70 and CD38, occult, including information that may inform ontime to progression, progression free survival, overall survival, orevent-free survival.

In some embodiments of the methods of the invention, classifying thecell based on activation levels of activatable element includesclassifying the cell as a cell that is correlated to a patient responseto a treatment, such as complete response, partial response, nodularpartial response, no response, progressive disease, stable disease,relapse or adverse reaction. The method may further comprise determininga method of treatment, e.g., chemotherapy, biological therapy, radiationtherapy, bone marrow transplantation, Peripheral stem celltransplantation, umbilical cord blood transplantation, autologous stemcell transplantation, allogeneic stem cell transplantation, syngeneicstem cell transplantation, surgery, induction therapy, maintenancetherapy, watchful waiting, or holistic/alternative therapy.

In some embodiments of the methods of the invention, the classifying ofthe cell based on activation level includes classifying the cell as acell that is correlated with minimal residual disease or emergingresistance.

In some embodiments of the invention, the activation level of theplurality of activatable elements in the cell is selected from the groupconsisting of cleavage by extracellular or intracellular proteaseexposure, novel hetero-oligomer formation, glycosylation level,phosphorylation level, acetylation level, methylation level,biotinylation level, glutamylation level, glycylation level,hydroxylation level, isomerization level, prenylation level,myristoylation level, lipoylation level, phosphopantetheinylation level,sulfation level, ISGylation level, nitrosylation level, palmitoylationlevel, SUMOylation level, ubiquitination level, neddylation level,citrullination level, deamidation level, disulfide bond formation level,proteolytic cleavage level, translocation level, changes in proteinturnover, multi-protein complex level, oxidation level, multi-lipidcomplex, and biochemical changes in cell membrane. In some embodiments,the activation level is a phosphorylation level. In some embodiments,the activatable element is selected from the group consisting ofproteins, carbohydrates, lipids, nucleic acids and metabolites. In someembodiments, the activatable element is a protein. In some embodiments,the activatable element is a change in metabolic state, temperature, orlocal ion concentration. In embodiments where the activatable element isa protein, in some embodiments the protein is a protein subject tophosphorylation or dephosphorylation, such as kinases, phosphatases,adaptor/scaffold proteins, ubiquitination enzymes, adhesion molecules,contractile proteins, cytoskeletal proteins, heterotrimeric G proteins,small molecular weight GTPases, guanine nucleotide exchange factors,GTPase activating proteins, caspases and proteins involved in apoptosis(e.g. PARP), ion channels, molecular transporters, molecular chaperones,metabolic enzymes, vesicular transport proteins, hydroxylases,isomerases, transferases, deacetylases, methylases, demethylases,proteases, esterases, hydrolases, DNA binding proteins or transcriptionfactors. In some embodiments, the protein is selected from the groupconsisting of PI3-Kinase (p85, p110a, p110b, p110d), Jak1, Jak2, SOCs,Rac, Rho, Cdc42, Ras-GAP, Vav, Tiam, Sos, Dbl, Nck, Gab, PRK, SHPT, andSHP2, SHIP1, SHIP2, sSHIP, PTEN, Shc, Grb2, PDK1, SGK, Akt1, Akt2, Akt3,TSC1,2, Rheb, mTor, 4EBP-1, p70S6Kinase, S6, LKB-1, AMPK, PFK,Acetyl-CoAa Carboxylase, DokS, Rafs, Mos, Tp12, MEK1/2, MLK3, TAK, DLK,MKK3/6, MEKK1,4, MLK3, ASK1, MKK4/7, SAPK/JNK1,2,3, p38s, Erk1/2, Syk,Btk, BLNK, LAT, ZAP70, Lck, Cbl, SLP-76, PLCγ1, PLCγ2, STAT1, STAT 3,STAT 4, STAT 5, STAT 6, FAK, p130CAS, PAKs, LIMK1/2, Hsp90, Hsp70,Hsp27, SMADs, Rel-A (p65-NFKB), CREB, Histone H2B, HATs, HDACs, PKR, Rb,Cyclin D, Cyclin E, Cyclin A, Cyclin B, P16, p14Arf, p27KIP, p21CIP,Cdk4, Cdk6, Cdk7, Cdk1, Cdk2, Cdk9, Cdc25,A/B/C, Abl, E2F, FADD, TRADD,TRAF2, RIP, Myd88, BAD, Bcl-2, Mcl-1, Bcl-XL, Caspase 2, Caspase 3,Caspase 6, Caspase 7, Caspase 8, Caspase 9, PARP, IAPB, Smac, Fodrin,Actin, Src, Lyn, Fyn, Lck, NIK, IκB, p65(RelA), IKKα, PKA, PKCα, PKCβ,PKCθ, PKCδ, CAMK, Elk, AFT, Myc, Egr-1, NFAT, ATF-2, Mdm2, p53, DNA-PK,Chk1, Chk2, ATM, ATR, β-catenin, CrkL, GSK3α, GSK3β, and FOXO. In someembodiments, the protein selected from the group consisting of Erk, Syk,Zap70, Lck, Btk, BLNK, Cbl, PLCγ₂, Akt, RelA, p38, S6. In someembodiments the protein is S6.

In some embodiments, the protein is selected from the group consistingof HER receptors, PDGF receptors, Kit receptor, FGF receptors, Ephreceptors, Trk receptors, IGF receptors, Insulin receptor, Met receptor,Ret, VEGF receptors, TIE1, TIE2, FAK, Jak1, Jak2, Jak3, Tyk2, Src, Lyn,Fyn, Lck, Fgr, Yes, Csk, Abl, Btk, ZAP70, Syk, IRAKs, cRaf, ARaf, BRAF,Mos, Lim kinase, ILK, Tpl, ALK, TGFIβ receptors, BMP receptors, MEKKs,ASK, MLKs, DLK, PAKs, Mek 1, Mek 2, MKK3/6, MKK4/7, ASK1, Cot, NIK, Bub,Myt 1, Weel, Casein kinases, PDK1, SGK1, SGK2, SGK3, Akt1, Akt2, Akt3,p90Rsks, p70S6Kinase, Prks, PKCs, PKAs, ROCK 1, ROCK 2, Auroras, CaMKs,MNKs, AMPKs, MELK, MARKs, Chk1, Chk2, LKB-1, MAPKAPKs, Pim1, Pim2, Pim3,IKKs, Cdks, Jnks, Erks, IKKs, GSK3α, GSK3β, Cdks, CLKs, PKR, PI3-Kinaseclass 1, class 2, class 3, mTor, SAPK/JNK1,2,3, p38s, PKR, DNA-PK, ATM,ATR, Receptor protein tyrosine phosphatases (RPTPs), LAR phosphatase,CD45, Non receptor tyrosine phosphatases (NPRTPs), SHPs, MAP kinasephosphatases (MKPs), Dual Specificity phosphatases (DUSPs), CDC25phosphatases, Low molecular weight tyrosine phosphatase, Eyes absent(EYA) tyrosine phosphatases, Slingshot phosphatases (SSH), serinephosphatases, PP2A, PP2B, PP2C, PP1, PP5, inositol phosphatases, PTEN,SHIPs, myotubularins, phosphoinositide kinases, phospholipases,prostaglandin synthases, 5-lipoxygenase, sphingosine kinases,sphingomyelinases, adaptor/scaffold proteins, Shc, Grb2, BLNK, LAT, Bcell adaptor for PI3-kinase (BCAP), SLAP, Dok, KSR, MyD88, Crk, CrkL,GAD, Nck, Grb2 associated binder (GAB), Fas associated death domain(FADD), TRADD, TRAF2, RIP, T-Cell leukemia family, IL-2, IL-4, IL-8,IL-6, interferon γ, interferon a, suppressors of cytokine signaling(SOCs), Cbl, SCF ubiquitination ligase complex, APC/C, adhesionmolecules, integrins, Immunoglobulin-like adhesion molecules, selectins,cadherins, catenins, focal adhesion kinase, p130CAS, fodrin, actin,paxillin, myosin, myosin binding proteins, tubulin, eg5/KSP, CENPs,β-adrenergic receptors, muscarinic receptors, adenylyl cyclasereceptors, small molecular weight GTPases, H-Ras, K-Ras, N-Ras, Ran,Rac, Rho, Cdc42, Arfs, RABs, RHEB, Vav, Tiam, Sos, Dbl, PRK, TSC1,2,Ras-GAP, Arf-GAPs, Rho-GAPs, caspases, Caspase 2, Caspase 3, Caspase 6,Caspase 7, Caspase 8, Caspase 9, PARP, Bcl-2, Mcl-1, Bcl-XL, Bcl-w,Bcl-B, A1, Bax, Bak, Bok, Bik, Bad, Bid, Bim, Bmf, Hrk, Noxa, Puma,IAPB, XIAP, Smac, Cdk4, Cdk 6, Cdk 2, Cdk1, Cdk 7, Cyclin D, Cyclin E,Cyclin A, Cyclin B, Rb, p16, p14Arf, p27KIP, p21CIP, molecularchaperones, Hsp90s, Hsp70, Hsp27, metabolic enzymes, Acetyl-CoAaCarboxylase, ATP citrate lyase, nitric oxide synthase, caveolins,endosomal sorting complex required for transport (ESCRT) proteins,vesicular protein sorting (Vsps), hydroxylases, prolyl-hydroxylasesPHD-1, 2 and 3, asparagine hydroxylase FIH transferases, Pin1 prolylisomerase, topoisomerases, deacetylases, Histone deacetylases, sirtuins,histone acetylases, CBP/P300 family, MYST family, ATF2, DNA methyltransferases, Histone H3K4 demethylases, H3K27, JHDM2A, UTX, VHL, WT-1,p53, Hdm, PTEN, ubiquitin proteases, urokinase-type plasminogenactivator (uPA) and uPA receptor (uPAR) system, cathepsins,metalloproteinases, esterases, hydrolases, separase, potassium channels,sodium channels, multi-drug resistance proteins, P-Gycoprotein,nucleoside transporters, Ets, Elk, SMADs, Rel-A (p65-NFκB), CREB, NFAT,ATF-2, AFT, Myc, Fos, Sp1, Egr-1, T-bet, β-catenin, HIFs, FOXOs, E2Fs,SRFs, TCFs, Egr-1, β-catenin, FOXO STAT1, STAT 3, STAT 4, STAT 5, STAT6, p53, WT-1, HMGA, pS6, 4EPB-1, eIF4E-binding protein, RNA polymerase,initiation factors, elongation factors.

In some embodiments, the invention provides methods for determining thepresence or absence of a condition in an individual by subjecting a cellfrom the individual to a modulator or an inhibitor, determining theactivation level of an activatable element in the cell and determiningthe presence or absence of the condition based on the activation level.In some embodiments, the cell is a hematopoietic derived cell. In someembodiments, the hematopoietically derived cell is selected from thegroup consisting of pluripotent hematopoietic stem cells, B-lymphocytelineage progenitor or derived cells, T-lymphocyte lineage progenitor orderived cells, NK cell lineage progenitor or derived cells, granulocytelineage progenitor or derived cells, monocyte lineage progenitor orderived cells, megakaryocyte lineage progenitor or derived cells anderythroid lineage progenitor or derived cells. In some embodiments, thehematopoietic derived cell is a B-lymphocyte lineage progenitor andderived cell, e.g., an early pro-B cell, late pro-B cell, large pre-Bcell, small pre-B cell, immature B cell, mature B cell, plasma cell andmemory B cell, a CD5+ B cell, a CD38+ B cell, a B cell bearing amutatated or non mutated heavy chain of the B cell receptor, or a B cellexpressing Zap70. In some embodiments, the condition is a neoplastic orhematopoietic condition.

In some embodiments of the methods of the invention, the modulator towhich the cell is subjected is an activator or an inhibitor. In someembodiments, the modulator is, e.g., a growth factor, cytokine, adhesionmolecule modulator, hormone, small molecule, polynucleotide, antibodies,natural compounds, lactones, chemotherapeutic agents, immune modulator,carbohydrate, proteases, ions, reactive oxygen species, or radiation. Insome embodiments, the modulator is a B cell receptor modulator, e.g., aB cell receptor activator such as a cross-linker of the B cell receptorcomplex or the B-cell co-receptor complex. In some embodiments, thecross-linker is an antibody, or molecular binding entity. In someembodiments, the cross-linker is an antibody, such as a multivalentantibody. In some embodiments, the antibody is a monovalent, bivalent,or multivalent antibody made more multivalent by attachment to a solidsurface or tethered on a nanoparticle surface to increase the localvalency of the epitope binding domain. In some embodiments, thecross-linker is a molecular binding entity, such as an entity that actsupon or binds the B cell receptor complex via carbohydrates or anepitope in the complex. In some embodiments, the molecular bindingentity is a monovalent, bivalent, or multivalent binding entity that ismade more multivalent by attachment to a solid surface or tethered on ananoparticle surface to increase the local valency of the epitopebinding domain. In some embodiments where the modulator is a B cellreceptor modulator, e.g., a B cell receptor activator such as across-linker of the B cell receptor complex or the B-cell co-receptorcomplex, cross-linking includes binding of an antibody or molecularbinding entity to the cell and then causing its crosslinking viainteraction of the cell with a solid surface that causes crosslinking ofthe BCR complex via antibody or molecular binding entity. In someembodiments, the crosslinker is selected from the group consisting ofF(ab)2 IgM, IgG, IgD, polyclonal BCR antibodies, monoclonal BCRantibodies, Fc receptor derived binding elements. The Ig may be derivedfrom a species selected from the group consisting of mouse, goat,rabbit, pig, rat, horse, cow, shark, chicken, or llama. In someembodiments, the crosslinker is F(ab)2 IgM, Polyclonal IgM antibodies,Monoclonal IgM antibodies, Biotinylated F(ab)2 IgCM, BiotinylatedPolyclonal IgM antibodies, Biotinylated Monoclonal IgM antibodies and/ora combination thereof.

In some embodiments, the inhibitor is a kinase or phosphatase inhibitor,such as adaphostin, AG 490, AG 825, AG 957, AG 1024, aloisine, aloisineA, alsterpaullone, aminogenistein, API-2, apigenin, arctigenin,AY-22989, BAY 61-3606, bisindolylmaleimide IX, chelerythrine,10-[4′-(N,N-Diethylamino)butyl]-2-chlorophenoxazine hydrochloride,dasatinib, 2-Dimethylamino-4,5,6,7-tetrabromo-1H-benzimidazole,5,7-Dimethoxy-3-(4-pyridinyl)quinoline dihydrochloride, edelfosine,ellagic acid, enzastaurin, ER 27319 maleate, erlotinib, ET18OCH3,fasudil, flavopiridol, gefitinib, GW 5074, H-7, H-8, H-89, HA-100,HA-1004, HA-1077, HA-1100, hydroxyfasudil, indirubin-3′-oxime,5-Iodotubercidin, kenpaullone, KN-62, KY12420, LFM-A13, lavendustin A,luteolin, LY-294002, LY294002, mallotoxin, ML-9, NSC-154020, NSC-226080,NSC-231634, NSC-664704, NSC-680410, NU6102, olomoucine, oxindole I,PD-153035, PD-98059, PD-169316, phloretin, phloridzin, piceatannol,picropodophyllin, PK1, PP1, PP2, purvalanol A, quercetin, R406, R788,rapamune, rapamycin, Ro 31-8220, roscovitine, rottlerin, SB202190,SB203580, sirolimus, sorafenib, SL327, SP600125, staurosporine, STI-571,SU-11274, SU1498, SU4312, SU6656, 4,5,6,7-Tetrabromotriazole, TG101348,Triciribine, Tyrphostin AG 490, Tyrphostin AG 825, Tyrphostin AG 957,Tyrphostin AG 1024, Tyrphostin SU1498, U0126, VX-509, VX-667, VX-680,W-7, wortmannin, XL-019, XL-147, XL-184, XL-228, XL-281, XL-518, XL-647,XL-765, XL-820, XL-844, XL-880, Y-27632, ZD-1839, ZM-252868, ZM-447439,H₂O₂, siRNA, miRNA, Cantharidin, (−)-p-Bromotetramisole, Microcystin LR,Sodium Orthovanadate, Sodium Pervanadate, Vanadyl sulfate, Sodiumoxodiperoxo(1,10-phenanthroline)vanadate, bis(maltolato)oxovanadium(IV),Sodium Molybdate, Sodium Perm olybdate, Sodium Tartrate, Imidazole,Sodium Fluoride, β-Glycerophosphate, Sodium Pyrophosphate Decahydrate,Calyculin A, Discodermia calyx, bpV(phen), mpV(pic), DMHV, Cypermethrin,Dephostatin, Okadaic Acid, NIPP-1,N-(9,10-Dioxo-9,10-dihydro-phenanthren-2-yl)-2,2-dimethyl-propionamide,α-Bromo-4-hydroxyacetophenone, 4-Hydroxyphenacyl Br,α-Bromo-4-methoxyacetophenone, 4-Methoxyphenacyl Br,α-Bromo-4-(carboxymethoxy)acetophenone, 4-(Carboxymethoxy)phenacyl Br,and bis(4-Trifluoromethylsulfonamidophenyl)-1,4-diisopropylbenzene,phenyarsine oxide, Pyrrolidine Dithiocarbamate, or Aluminum fluoride. Insome embodiments the phosphatase inhibitor is H₂O₂.

In some embodiments of the methods of the invention, the cell issubjected to a B cell receptor activator and a phosphatase inhibitor orkinase inhibitor, such as F(ab)₂IgM or biotinylated F(ab)₂IgM and aphosphatase inhibitor (e.g. H₂O₂).

In some embodiments, the invention provides a method of determining atonic signaling status of a cell by subjecting the cell to a modulator,determining the activation level of an activatable element thatparticipates in a tonic signaling pathway in the cell, and determiningthe status of a tonic signaling pathway in the cell from the activationlevel. In some embodiments, a condition of an individual is determinedbased on tonic signaling status of a cell. In some embodiments, thecondition is a neoplastic and/or hematopoietic condition. In someembodiments, the neoplastic or hematopoietic condition is selected fromthe group consisting of Non-Hodgkin Lymphoma, Hodgkin or otherlymphomas, acute or chronic leukemias, polycythemias, thrombocythemias,multiple myeloma and plasma cell disorders, e.g., amyloidosis andWaldenstrom's macroglobulinemia, myelodysplastic disorders,myeloproliferative disorders, myelofibrosis, and atypical immunelymphoproliferations. In some embodiments, the neoplastic orhematopoietic condition is non-B lineage derived, such as acute myeloidleukemia (AML), Chronic Myeloid Leukemia (CML), non-B cell acutelymphocytic leukemia (ALL), non-B cell lymphomas, myelodysplasticdisorders, myeloproliferative disorders, myelofibrosis, polycythemias,thrombocythemias, and non-B atypical immune lymphoproliferations. Insome embodiments, the neoplastic or hematopoietic condition is a B-Cellor B cell lineage derived disorder such as B-Cell or B cell lineagederived disorder is selected from the group consisting of ChronicLymphocytic Leukemia (CLL), B lymphocyte lineage leukemia, B lymphocytelineage lymphoma, Multiple Myeloma, and plasma cell disorders, e.g.,amyloidosis and Waldenstrom's macroglobulinemia. In some embodiments,the condition is CLL. In some embodiments, the CLL is defined by amonoclonal B cell population that co-expresses CD5 with CD19 and CD23 orCD5 with CD20 and CD23 and by surface immunoglobulin expression.

In some embodiments, the tonic signaling status of a cell is correlatedwith a clinical outcome such as prognosis or diagnosis of the condition.In some embodiments, the clinical outcome is the staging or grading of aneoplastic or hematopoietic condition, such as aggressive, indolent,benign, refractory, Roman Numeral staging, TNM Staging, Rai staging,Binet staging, WHO classification, FAB classification, IPSS score, WPSSscore, limited stage, extensive stage, staging according to molecularmarkers such as ZAP70, the mutational status of the heavy chain of theB-cell receptor (IgVH) and CD38, occult, including information that mayinform on time to progression, progression free survival, overallsurvival, or event-free survival.

In some embodiments, the correlation is determining the individual'sresponse to a treatment, e.g., normal responder, hyper responder, poorresponder, having emerging resistance, non-compliant, and adversereaction.

In some embodiments, the correlation includes classifying the cell asminimal residual disease or emerging resistance. The correlation mayfurther include determining a method of treatment, such as chemotherapy,biological therapy, radiation therapy, bone marrow transplantation,Peripheral stem cell transplantation, umbilical cord bloodtransplantation, autologous stem cell transplantation, allogeneic stemcell transplantation, syngeneic stem cell transplantation, surgery,induction therapy, maintenance therapy, or watchful waiting.

In some embodiments of this aspect, the invention provides a method ofcorrelating an activation level of a B-lymphocyte lineage derived cellwith a neoplastic or hematopoietic condition in an individual bysubjecting the B-lymphocyte lineage derived cell from the individual toa modulator; determining the activation levels of a plurality ofactivatable elements that participate in a tonic signaling pathway inthe B-lymphocyte lineage derived cell; and identifying a pattern of theactivation levels of the plurality of activatable elements in the tonicsignaling pathway in the cell that correlates with a clinical outcome,such as the prediction of outcome for a particular treatment, aprognosis or diagnosis of a cetain condition (e.g. a neoplasticcondition). In some embodiments, the B-lymphocyte lineage progenitor orderived cell is selected from the group consisting of early pro-B cell,late pro-B cell, large pre-B cell, small pre-B cell, immature B cell,mature B cell, plasma cell and memory B cell, a CD5+B cell, a CD38+Bcell, a B cell bearing a mutatated or non mutated heavy chain of the Bcell receptor and a B cell expressing Zap70. In some embodiments, thecorrelation is determining a clinical outcome, such as prognosis ordiagnosis of the condition.

In some embodiments of the methods of the invention, the modulator towhich the cell is subjected is an activator or an inhibitor. In someembodiments, the modulator is, e.g., a growth factor, cytokine, adhesionmolecule modulator, hormone, small molecule, polynucleotide, antibodies,natural compounds, lactones, chemotherapeutic agents, immune modulator,carbohydrate, proteases, ions, reactive oxygen species, or radiation. Insome embodiments, the modulator is a B cell receptor modulator, e.g., aB cell receptor activator such as a cross-linker of the B cell receptorcomplex or the B-cell co-receptor complex. In some embodiments, thecross-linker is an antibody, or molecular binding entity. In someembodiments, the cross-linker is an antibody, such as a multivalentantibody. In some embodiments, the antibody is a monovalent, bivalent,or multivalent antibody made more multivalent by attachment to a solidsurface or tethered on a nanoparticle surface to increase the localvalency of the epitope binding domain. In some embodiments, thecross-linker is a molecular binding entity, such as an entity that actsupon or binds the B cell receptor complex via carbohydrates or anepitope in the complex. In some embodiments, the molecular bindingentity is a monovalent, bivalent, or multivalent binding entity that ismade more multivalent by attachment to a solid surface or tethered on ananoparticle surface to increase the local valency of the epitopebinding domain. In some embodiments where the modulator is a B cellreceptor modulator, e.g., a B cell receptor activator such as across-linker of the B cell receptor complex or the B-cell co-receptorcomplex, cross-linking includes binding of an antibody or molecularbinding entity to the cell and then causing its crosslinking viainteraction of the cell with a solid surface that causes crosslinking ofthe BCR complex via antibody or molecular binding entity. In someembodiments, the crosslinker is selected from the group consisting ofF(ab)2 IgM, IgG, IgD, polyclonal BCR antibodies, monoclonal BCRantibodies, Fc receptor derived binding elements and/or a combinationthereof. The Ig may be derived from a species selected from the groupconsisting of mouse, goat, rabbit, pig, rat, horse, cow, shark, chicken,or llama. In some embodiments, the crosslinker is F(ab)2 IgM, PolyclonalIgM antibodies, Monoclonal IgM antibodies, Biotinylated F(ab)2 IgCM,Biotinylated Polyclonal IgM antibodies, Biotinylated Monoclonal IgMantibodies and/or a combination thereof.

In some embodiments of the methods of the invention, the modulator towhich the cell is subjected is an inhibitor of a cellular factor or aplurality of factors that participates in a signaling cascade in thecell. In some embodiments, the inhibitor is a kinase or phosphataseinhibitor, such as adaphostin, AG 490, AG 825, AG 957, AG 1024,aloisine, aloisine A, alsterpaullone, aminogenistein, API-2, apigenin,arctigenin, AY-22989, BAY 61-3606, bisindolylmaleimide IX,chelerythrine, 10-[4′-(N,N-Diethylamino)butyl]-2-chlorophenoxazinehydrochloride, dasatinib,2-Dimethylamino-4,5,6,7-tetrabromo-1H-benzimidazole,5,7-Dimethoxy-3-(4-pyridinyl)quinoline dihydrochloride, edelfosine,ellagic acid, enzastaurin, ER 27319 maleate, erlotinib, ET18OCH3,fasudil, flavopiridol, gefitinib, GW 5074, H-7, H-8, H-89, HA-100,HA-1004, HA-1077, HA-1100, hydroxyfasudil, indirubin-3′-oxime,5-Iodotubercidin, kenpaullone, KN-62, KY12420, LFM-A13, lavendustin A,luteolin, LY-294002, LY294002, mallotoxin, ML-9, NSC-154020, NSC-226080,NSC-231634, NSC-664704, NSC-680410, NU6102, olomoucine, oxindole I,PD-153035, PD-98059, PD-169316, phloretin, phloridzin, piceatannol,picropodophyllin, PK1, PP1, PP2, purvalanol A, quercetin, R406, R788,rapamune, rapamycin, Ro 31-8220, roscovitine, rottlerin, SB202190,SB203580, sirolimus, sorafenib, SL327, SP600125, staurosporine, STI-571,SU-11274, SU1498, SU4312, SU6656, 4,5,6,7-Tetrabromotriazole, TG101348,Triciribine, Tyrphostin AG 490, Tyrphostin AG 825, Tyrphostin AG 957,Tyrphostin AG 1024, Tyrphostin SU1498, U0126, VX-509, VX-667, VX-680,W-7, wortmannin, XL-019, XL-147, XL-184, XL-228, XL-281, XL-518, XL-647,XL-765, XL-820, XL-844, XL-880, Y-27632, ZD-1839, ZM-252868, ZM-447439,H₂O₂, siRNA, miRNA, Cantharidin, (−)-p-Bromotetramisole, Microcystin LR,Sodium Orthovanadate, Sodium Pervanadate, Vanadyl sulfate, Sodiumoxodiperoxo(1,10-phenanthroline)vanadate, bis(maltolato)oxovanadium(IV),Sodium Molybdate, Sodium Perm olybdate, Sodium Tartrate, Imidazole,Sodium Fluoride, β-Glycerophosphate, Sodium Pyrophosphate Decahydrate,Calyculin A, Discodermia calyx, bpV(phen), mpV(pic), DMHV, Cypermethrin,Dephostatin, Okadaic Acid, NIPP-1,N-(9,10-Dioxo-9,10-dihydro-phenanthren-2-yl)-2,2-dimethyl-propionamide,α-Bromo-4-hydroxyacetophenone, 4-Hydroxyphenacyl Br,α-Bromo-4-methoxyacetophenone, 4-Methoxyphenacyl Br,α-Bromo-4-(carboxymethoxy)acetophenone, 4-(Carboxymethoxy)phenacyl Br,and bis(4-Trifluoromethylsulfonamidophenyl)-1,4-diisopropylbenzene,phenyarsine oxide, Pyrrolidine Dithiocarbamate, or Aluminum fluoride. Insome embodiments the phosphatase inhibitor is H₂O₂.

In some embodiments of the methods of the invention, the cell is furthersubjected to a second modulator, e.g., the cell may be subjected to a Bcell receptor activator and a phosphatase inhibitor, such as F(ab)₂IgMor biotinylated F(ab)₂IgM and a phosphatase inhibitor (e.g. H₂O₂).

In some embodiments of the invention, the activation level of theplurality of activatable elements in the cell is selected from the groupconsisting of cleavage by extracellular or intracellular proteaseexposure, novel hetero-oligomer formation, glycosylation level,phosphorylation level, acetylation level, methylation level,biotinylation level, glutamylation level, glycylation level,hydroxylation level, isomerization level, prenylation level,myristoylation level, lipoylation level, phosphopantetheinylation level,sulfation level, ISGylation level, nitrosylation level, palmitoylationlevel, SUMOylation level, ubiquitination level, neddylation level,citrullination level, deamidation level, disulfide bond formation level,proteolytic cleavage level, translocation level, changes in proteinturnover, multi-protein complex level, oxidation level, multi-lipidcomplex, and biochemical changes in cell membrane. In some embodiments,the activation level is a phosphorylation level. In some embodiments,the activatable element is selected from the group consisting ofproteins, carbohydrates, lipids, nucleic acids and metabolites. In someembodiments, the activatable element is a protein. In some embodiments,the activatable element is a change in metabolic state, temperature, orlocal ion concentration. In embodiments where the activatable element isa protein, in some embodiments the protein is a protein subject tophosphorylation or dephosphorylation, such as kinases, phosphatases,adaptor/scaffold proteins, ubiquitination enzymes, adhesion molecules,contractile proteins, cytoskeletal proteins, heterotrimeric G proteins,small molecular weight GTPases, guanine nucleotide exchange factors,GTPase activating proteins, caspases and proteins involved in apoptosis(e.g. PARP), ion channels, molecular transporters, molecular chaperones,metabolic enzymes, vesicular transport proteins, hydroxylases,isomerases, transferases, deacetylases, methylases, demethylases,proteases, esterases, hydrolases, DNA binding proteins or transcriptionfactors. In some embodiments, the protein is selected from the groupconsisting of PI3-Kinase (p85, p110a, p110b, p110d), Jak1, Jak2, SOCs,Rac, Rho, Cdc42, Ras-GAP, Vav, Tiam, Sos, Dbl, Nck, Gab, PRK, SHPT, andSHP2, SHIP1, SHIP2, sSHIP, PTEN, Shc, Grb2, PDK1, SGK, Akt1, Akt2, Akt3,TSC1,2, Rheb, mTor, 4EBP-1, p70S6Kinase, S6, LKB-1, AMPK, PFK,Acetyl-CoAa Carboxylase, DokS, Rafs, Mos, Tp12, MEK1/2, MLK3, TAK, DLK,MKK3/6, MEKK1,4, MLK3, ASK1, MKK4/7, SAPK/JNK1,2,3, p38s, Erk1/2, Syk,Btk, BLNK, LAT, ZAP70, Lck, Cbl, SLP-76, PLCγ₁, PLCγ₂, STAT1, STAT 3,STAT 4, STAT 5, STAT 6, FAK, p130CAS, PAKs, LIMK1/2, Hsp90, Hsp70,Hsp27, SMADs, Rel-A (p65-NFKB), CREB, Histone H2B, HATs, HDACs, PKR, Rb,Cyclin D, Cyclin E, Cyclin A, Cyclin B, P16, p14Arf, p27KIP, p21CIP,Cdk4, Cdk6, Cdk7, Cdk1, Cdk2, Cdk9, Cdc25,A/B/C, Abl, E2F, FADD, TRADD,TRAF2, RIP, Myd88, BAD, Bcl-2, Mcl-1, Bcl-XL, Caspase 2, Caspase 3,Caspase 6, Caspase 7, Caspase 8, Caspase 9, PARP, IAPB, Smac, Fodrin,Actin, Src, Lyn, Fyn, Lck, NIK, IκB, p65(RelA), IKKα, PKA, PKCα, PKCβ,PKCθ, PKCδ, CAMK, Elk, AFT, Myc, Egr-1, NFAT, ATF-2, Mdm2, p53, DNA-PK,Chk1, Chk2, ATM, ATR, β-catenin, CrkL, GSK3α, GSK3β, and FOXO. In someembodiments, the protein selected from the group consisting of Erk, Syk,Zap70, Lck, Btk, BLNK, Cbl, PLCγ₂, Akt, RelA, p38, S6. In someembodiments the protein is S6.

In some embodiments, the protein is selected from the group consistingof HER receptors, PDGF receptors, Kit receptor, FGF receptors, Ephreceptors, Trk receptors, IGF receptors, Insulin receptor, Met receptor,Ret, VEGF receptors, TIE1, TIE2, FAK, Jak1, Jak2, Jak3, Tyk2, Src, Lyn,Fyn, Lck, Fgr, Yes, Csk, Abl, Btk, ZAP70, Syk, IRAKs, cRaf, ARaf, BRAF,Mos, Lim kinase, ILK, Tpl, ALK, TGFβ receptors, BMP receptors, MEKKs,ASK, MLKs, DLK, PAKs, Mek 1, Mek 2, MKK3/6, MKK4/7, ASK1, Cot, NIK, Bub,Myt 1, Weel, Casein kinases, PDK1, SGK1, SGK2, SGK3, Akt1, Akt2, Akt3,p90Rsks, p70S6Kinase, Prks, PKCs, PKAs, ROCK 1, ROCK 2, Auroras, CaMKs,MNKs, AMPKs, MELK, MARKs, Chk1, Chk2, LKB-1, MAPKAPKs, Pim 1, Pim2,Pim3, IKKs, Cdks, Jnks, Erks, IKKs, GSK3α, GSK3β, Cdks, CLKs, PKR,PI3-Kinase class 1, class 2, class 3, mTor, SAPK/JNK1,2,3, p38s, PKR,DNA-PK, ATM, ATR, Receptor protein tyrosine phosphatases (RPTPs), LARphosphatase, CD45, Non receptor tyrosine phosphatases (NPRTPs), SHPs,MAP kinase phosphatases (MKPs), Dual Specificity phosphatases (DUSPs),CDC25 phosphatases, Low molecular weight tyrosine phosphatase, Eyesabsent (EYA) tyrosine phosphatases, Slingshot phosphatases (SSH), serinephosphatases, PP2A, PP2B, PP2C, PP1, PP5, inositol phosphatases, PTEN,SHIPs, myotubularins, phosphoinositide kinases, phospholipases,prostaglandin synthases, 5-lipoxygenase, sphingosine kinases,sphingomyelinases, adaptor/scaffold proteins, Shc, Grb2, BLNK, LAT, Bcell adaptor for PI3-kinase (BCAP), SLAP, Dok, KSR, MyD88, Crk, CrkL,GAD, Nck, Grb2 associated binder (GAB), Fas associated death domain(FADD), TRADD, TRAF2, RIP, T-Cell leukemia family, IL-2, IL-4, IL-8,IL-6, interferon γ, interferon α, suppressors of cytokine signaling(SOCs), Cbl, SCF ubiquitination ligase complex, APC/C, adhesionmolecules, integrins, Immunoglobulin-like adhesion molecules, selectins,cadherins, catenins, focal adhesion kinase, p130CAS, fodrin, actin,paxillin, myosin, myosin binding proteins, tubulin, eg5/KSP, CENPs,β-adrenergic receptors, muscarinic receptors, adenylyl cyclasereceptors, small molecular weight GTPases, H-Ras, K-Ras, N-Ras, Ran,Rac, Rho, Cdc42, Arfs, RABs, RHEB, Vav, Tiam, Sos, Dbl, PRK, TSC1,2,Ras-GAP, Arf-GAPs, Rho-GAPs, caspases, Caspase 2, Caspase 3, Caspase 6,Caspase 7, Caspase 8, Caspase 9, PARP, Bcl-2, Mcl-1, Bcl-XL, Bcl-w,Bcl-B, A1, Bax, Bak, Bok, Bik, Bad, Bid, Bim, Bmf, Hrk, Noxa, Puma,IAPB, XIAP, Smac, Cdk4, Cdk 6, Cdk 2, Cdk1, Cdk 7, Cyclin D, Cyclin E,Cyclin A, Cyclin B, Rb, p16, p14Arf, p27KIP, p21CIP, molecularchaperones, Hsp90s, Hsp70, Hsp27, metabolic enzymes, Acetyl-CoAaCarboxylase, ATP citrate lyase, nitric oxide synthase, caveolins,endosomal sorting complex required for transport (ESCRT) proteins,vesicular protein sorting (Vsps), hydroxylases, prolyl-hydroxylasesPHD-1, 2 and 3, asparagine hydroxylase FIH transferases, Pin1 prolylisomerase, topoisomerases, deacetylases, Histone deacetylases, sirtuins,histone acetylases, CBP/P300 family, MYST family, ATF2, DNA methyltransferases, Histone H3K4 demethylases, H3K27, JHDM2A, UTX, VHL, WT-1,p53, Hdm, PTEN, ubiquitin proteases, urokinase-type plasminogenactivator (uPA) and uPA receptor (uPAR) system, cathepsins,metalloproteinases, esterases, hydrolases, separase, potassium channels,sodium channels, multi-drug resistance proteins, P-Gycoprotein,nucleoside transporters, Ets, Elk, SMADs, Rel-A (p65-NFKB), CREB, NFAT,ATF-2, AFT, Myc, Fos, Sp1, Egr-1, T-bet, β-catenin, HIFs, FOXOs, E2Fs,SRFs, TCFs, Egr-1, β-catenin, FOXO STAT1, STAT 3, STAT 4, STAT 5, STAT6, p53, WT-1, HMGA, pS6, 4EPB-1, eIF4E-binding protein, RNA polymerase,initiation factors, elongation factors.

In addition to determining the activation level of an activatableprotein, in some embodiments the methods for classifying a cell furthercomprise determining the level of an additional intracellular markerand/or a cell surface marker. In some embodiments the methods forclassifying a cell comprise determining the level of an additionalintracellular marker. In some embodiments the intracelluar marker is acaptured intracellular cytokine. In some embodiments the methods forclassifying a cell comprise determining the level of an additional cellsurface marker. In some embodiments the cell surface marker is a cellsurface ligand or receptor. In some embodiments the cell surface markeris a component of a B-cell receptor. In some embodiments the cellsurface marker is CD45, CD5, CD19, CD20, CD22, CD23, CD27, CD37, CD40,CD52, CD79, CD38, CD96, major histocompatability antigen (MHC) Class1 orMHC Class 2.

In some embodiments the methods of the invention for prognosis,diagnosis, or determination of treatment further comprise determiningthe level of an additionalserum marker. In some embodiments the serummarker comprises a protein. In some embodiments the serum marker is acytokine, growth factor, chemokine, soluble receptor, small compound, orphamaceutical drug. In some embodiments the serum marker comprises acomponent or product of a pathogen or parasite. In some embodiment theserum marker is selected from a group consisting ofbeta-2-microglobulin, calcitonin, thymidine kinase and ferritin.

In some embodiments, the invention provides a method of correlating anactivation level of B-lymphocyte lineage derived cells with a neoplasticor hematopoietic condition in an individual by subjecting theB-lymphocyte lineage derived cell from the individual to a modulator;determining the activation levels of a plurality of activatable elementsin the B-lymphocyte lineage derived cell; and identifying a pattern ofthe activation levels of the plurality of activatable elements in thecell that correlates with the neoplastic condition. In some embodiments,the activatable element is selected from the group consisting ofelements selected from the group consisting of Erk, Syk, Zap70, Lck,Btk, BLNK, Cbl, PLCγ₂, Akt, RelA, p38, S6. In some embodiments, theactivatable element is selected from the group consisting of Cbl, PLCγ₂,and S6. In some embodiments, the activatable element is S6. In someembodiments, the B-lymphocyte lineage progenitor or derived cell isselected from the group consisting of early pro-B cell, late pro-B cell,large pre-B cell, small pre-B cell, immature B cell, mature B cell,plasma cell and memory B cell, a CD5+ B cell, a CD38+ B cell, a B cellbearing a mutatated or non mutated heavy chain of the B cell receptor,or a B cell expressing Zap70. In some embodiments, the inventionprovides methods for correlating and/or classifying an activation stateof a CLL cell with a clinical outcome in an individual by subjecting theCLL cell from the individual to a modulator, where the CLL cellexpresses a B-Cell receptor (BCR), determining the activation levels ofa plurality of activatable elements, and identifying a pattern of theactivation levels of the plurality of activatable elements to determinethe presence or absence of an alteration in signaling proximal to theBCR, wherein the presence of the alteration is indicative of a clinicaloutcome.

In some embodiments the method comprises identifying a pattern of saidactivation levels of said pluralilty of activatable elements in saidcell, wherein said pattern is correlated to a disease or condition.

In some embodiments, the correlation is determining a clinical outcome,such as prognosis or determination of treatment of the condition. Insome embodiments, the neoplastic or hematopoietic condition is selectedfrom the group consisting of Non-Hodgkin Lymphoma, Hodgkin or otherlymphomas, acute or chronic leukemias, polycythemias, thrombocythemias,multiple myeloma and plasma cell disorders, such as amyloidosis andWaldenstrom's macroglobulinemia, myelodysplastic disorders,myeloproliferative disorders, myelofibrosis, or atypical immunelymphoproliferations. In some embodiments, the neoplastic orhematopoietic condition is non-B lineage derived, such as acute myeloidleukemia (AML), Chronic Myeloid Leukemia (CML), non-B cell acutelymphocytic leukemia (ALL), non-B cell lymphomas, myelodysplasticdisorders, myeloproliferative disorders, myelofibrosis, polycythemias,thrombocythemias, and non-B atypical immune lymphoproliferations. Insome embodiments, the neoplastic or hematopoietic condition is a B-Cellor B cell lineage derived disorder, such as B-Cell or B cell lineagederived disorder is selected from the group consisting of ChronicLymphocytic Leukemia (CLL), B lymphocyte lineage leukemia, B lymphocytelineage lymphoma, Multiple Myeloma, or plasma cell disorders, e.g.,amyloidosis and Waldenstrom's macroglobulinemias. In some embodiments,the condition is CLL. In some embodiments the CLL is defined by amonoclonal B cell population that co-expresses CD5 with CD19 and CD23 orCD5 with CD20 and CD23 and dim surface immunoglobulin expression.

In some embodiments, the clinical outcome is the staging or grading of aneoplastic or hematopoietic condition, such as aggressive, indolent,benign, refractory, Roman Numeral staging, TNM Staging, Rai staging,Binet staging, WHO classification, FAB classification, IPSS score, WPSSscore, limited stage, extensive stage, staging according to cellularmarkers such as ZAP70, IgV_(H) mutational status and CD38, occult,including information that may inform on time to progression,progression free survival, overall survival, or event-free survival.

In some embodiments, the correlation is determining the individual'sresponse to a specific treatment, e.g., normal responder, hyperresponder, poor responder, having emerging resistance, non-compliant,and adverse reaction.

In some embodiments, the correlation includes classifying the cell asminimal residual disease or emerging resistance. The correlation mayfurther include determining a method of treatment, such as chemotherapy,biological therapy, targeted therapy, radiation therapy, bone marrowtransplantation, Peripheral stem cell transplantation, umbilical cordblood transplantation, autologous stem cell transplantation, allogeneicstem cell transplantation, syngeneic stem cell transplantation, surgery,induction therapy, maintenance therapy, or watchful waiting. Inadditional embodiments the correlation may further include determinationof the appropriate dosage or timing of a given treatment.

In some embodiments of the methods of the invention, the modulator towhich the cell is subjected is an activator or an inhibitor. In someembodiments, the modulator is, e.g., a growth factor, cytokine, adhesionmolecule modulator, hormone, small molecule, polynucleotide, antibodies,natural compounds, lactones, chemotherapeutic agents, immune modulator,carbohydrate, proteases, ions, reactive oxygen species, or radiation. Insome embodiments the modulator is an antibody e.g. anti-CD20 (Rituxan),anti-CD22 (epratuzumab), anti-CD23 (lumiliximab) or anti-CD52(Alemtuzumab), that recognize antigens on the cell surface. In someembodiments, the modulator is a B cell receptor complex modulator, e.g.,anti-CD20, which recognizes a component of the B cell receptorco-complex, or a B cell receptor activator such as a cross-linker of theB cell receptor complex or the B-cell co-receptor complex. In someembodiments, the cross-linker is an antibody, or molecular bindingentity. In some embodiments, the cross-linker is an antibody, such as amultivalent antibody. In some embodiments, the antibody is a monovalent,bivalent, or multivalent antibody made more multivalent by attachment toa solid surface or tethered on a nanoparticle surface to increase thelocal valency of the epitope binding domain. In some embodiments, thecross-linker is a molecular binding entity, such as an entity that actsupon or binds the B cell receptor complex via carbohydrates or anepitope in the complex. In some embodiments, the molecular bindingentity is a monovalent, bivalent, or multivalent binding entity that ismade more multivalent by attachment to a solid surface or tethered on ananoparticle surface to increase the local valency of the epitopebinding domain. In some embodiments where the modulator is a B cellreceptor modulator, e.g., a B cell receptor activator such as across-linker of the B cell receptor complex or the B-cell co-receptorcomplex, cross-linking includes binding of an antibody or molecularbinding entity to the cell and then causing its crosslinking viainteraction of the cell with a solid surface that causes crosslinking ofthe BCR complex via antibody or molecular binding entity. In someembodiments, the crosslinker is selected from the group consisting ofF(ab)2 IgM, IgG, IgD, polyclonal BCR antibodies, monoclonal BCRantibodies, Fc receptor derived binding elements and/or a combinationthereof. The Ig may be derived from a species selected from the groupconsisting of mouse, goat, rabbit, pig, rat, horse, cow, shark, chicken,or llama. In some embodiments, the crosslinker is F(ab)2 IgM, PolyclonalIgM antibodies, Monoclonal IgM antibodies, Biotinylated F(ab)2 IgCM,Biotinylated Polyclonal IgM antibodies, Biotinylated Monoclonal IgMantibodies and/or a combination thereof.

In some embodiments of the methods of the invention, the modulator towhich the cell is subjected is an inhibitor of a cellular factor or aplurality of factors that participates in a signaling cascade in thecell. In some embodiments, the inhibitor is a kinase or phosphataseinhibitor, such as adaphostin, AG 490, AG 825, AG 957, AG 1024,aloisine, aloisine A, alsterpaullone, aminogenistein, API-2, apigenin,arctigenin, AY-22989, BAY 61-3606, bisindolylmaleimide IX,chelerythrine, 10-[4′-(N,N-Diethylamino)butyl]-2-chlorophenoxazinehydrochloride, dasatinib,2-Dimethylamino-4,5,6,7-tetrabromo-1H-benzimidazole,5,7-Dimethoxy-3-(4-pyridinyl)quinoline dihydrochloride, edelfosine,ellagic acid, enzastaurin, ER 27319 maleate, erlotinib, ET18OCH3,fasudil, flavopiridol, gefitinib, GW 5074, H-7, H-8, H-89, HA-100,HA-1004, HA-1077, HA-1100, hydroxyfasudil, indirubin-3′-oxime,5-Iodotubercidin, kenpaullone, KN-62, KY12420, LFM-A13, lavendustin A,luteolin, LY-294002, LY294002, mallotoxin, ML-9, NSC-154020, NSC-226080,NSC-231634, NSC-664704, NSC-680410, NU6102, olomoucine, oxindole I,PD-153035, PD-98059, PD-169316, phloretin, phloridzin, piceatannol,picropodophyllin, PK1, PP1, PP2, purvalanol A, quercetin, R406, R788,rapamune, rapamycin, Ro 31-8220, roscovitine, rottlerin, SB202190,SB203580, sirolimus, sorafenib, SL327, SP600125, staurosporine, STI-571,SU-11274, SU1498, SU4312, SU6656, 4,5,6,7-Tetrabromotriazole, TG101348,Triciribine, Tyrphostin AG 490, Tyrphostin AG 825, Tyrphostin AG 957,Tyrphostin AG 1024, Tyrphostin SU1498, U0126, VX-509, VX-667, VX-680,W-7, wortmannin, XL-019, XL-147, XL-184, XL-228, XL-281, XL-518, XL-647,XL-765, XL-820, XL-844, XL-880, Y-27632, ZD-1839, ZM-252868, ZM-447439,H₂O₂, siRNA, miRNA, Cantharidin, (−)-p-Bromotetramisole, Microcystin LR,Sodium Orthovanadate, Sodium Pervanadate, Vanadyl sulfate, Sodiumoxodiperoxo(1,10-phenanthroline)vanadate, bis(maltolato)oxovanadium(IV),Sodium Molybdate, Sodium Perm olybdate, Sodium Tartrate, Imidazole,Sodium Fluoride, β-Glycerophosphate, Sodium Pyrophosphate Decahydrate,Calyculin A, Discodermia calyx, bpV(phen), mpV(pic), DMHV, Cypermethrin,Dephostatin, Okadaic Acid, NIPP-1,N-(9,10-Dioxo-9,10-dihydro-phenanthren-2-yl)-2,2-dimethyl-propionamide,α-Bromo-4-hydroxyacetophenone, 4-Hydroxyphenacyl Br,α-Bromo-4-methoxyacetophenone, 4-Methoxyphenacyl Br,α-Bromo-4-(carboxymethoxy)acetophenone, 4-(Carboxymethoxy)phenacyl Br,and bis(4-Trifluoromethylsulfonamidophenyl)-1,4-diisopropylbenzene,phenyarsine oxide, Pyrrolidine Dithiocarbamate, or Aluminum fluoride. Insome embodiments the phosphatase inhibitor is H₂O₂.

In some embodiments of the methods of the invention, the cell is furthersubjected to a second modulator, e.g., the cell may be subjected to a Bcell receptor activator and a kinase inhibitor or a phosphataseinhibitor, such as F(ab)₂IgM or biotinylated F(ab)₂IgM and H₂O₂.

In some embodiments of the invention, the activation level of theactivatable element in the cell is selected from the group consisting ofcleavage by extracellular or intracellular protease exposure, novelhetero-oligomer formation, glycosylation level, phosphorylation level,acetylation level, methylation level, biotinylation level, glutamylationlevel, glycylation level, hydroxylation level, isomerization level,prenylation level, myristoylation level, lipoylation level,phosphopantetheinylation level, sulfation level, ISGylation level,nitrosylation level, palmitoylation level, SUMOylation level,ubiquitination level, neddylation level, citrullination level,deamidation level, disulfide bond formation level, proteolytic cleavagelevel, translocation level, changes in protein turnover, multi-proteincomplex level, oxidation level, multi-lipid complex, and biochemicalchanges in cell membrane. In some embodiments, the activation level is aphosphorylation level. In some embodiments, the activatable element isselected from the group consisting of proteins, carbohydrates, lipids,nucleic acids and metabolites. In some embodiments, the activatableelement is a protein. In some embodiments, the activatable element is achange in metabolic state, temperature, or local ion concentration. Inembodiments where the activatable element is a protein, in someembodiments the protein is a protein subject to phosphorylation ordephosphorylation, such as kinases, phosphatases, adaptor/scaffoldproteins, ubiquitination enzymes, adhesion molecules, contractileproteins, cytoskeletal proteins, heterotrimeric G proteins, smallmolecular weight GTPases, guanine nucleotide exchange factors, GTPaseactivating proteins, caspases and proteins involved in apoptosis (e.g.PARP), ion channels, molecular transporters, molecular chaperones,metabolic enzymes, vesicular transport proteins, hydroxylases,isomerases, transferases, deacetylases, methylases, demethylases,proteases, esterases, hydrolases, DNA binding proteins or transcriptionfactors.

In another aspect, the invention provides for methods for determining aphenotypic profile of a population of cells by exposing the populationof cells to a plurality of modulators in separate cultures, where atleast one of the modulators is an inhibitor, determining the presence orabsence of an increase in activation level of an activatable element inthe cell population from each of the separate culture and classifyingthe cell population based on the presence or absence of the increase inthe activation of the activatable element from each of the separateculture. In some embodiments, the inhibitor is an inhibitor of acellular factor or a plurality of factors that participates in asignaling cascade in the cell. In some embodiments, the inhibitor is aphosphatase or kinase inhibitor. Examples of kinase inhibitors includeadaphostin, AG 490, AG 825, AG 957, AG 1024, aloisine, aloisine A,alsterpaullone, aminogenistein, API-2, apigenin, arctigenin, AY-22989,BAY 61-3606, bisindolylmaleimide IX, chelerythrine,10-[4′-(N,N-Diethylamino)butyl]-2-chlorophenoxazine hydrochloride,dasatinib, 2-Dimethylamino-4,5,6,7-tetrabromo-1H-benzimidazole,5,7-Dimethoxy-3-(4-pyridinyl)quinoline dihydrochloride, edelfosine,ellagic acid, enzastaurin, ER 27319 maleate, erlotinib, ET18OCH3,fasudil, flavopiridol, gefitinib, GW 5074, H-7, H-8, H-89, HA-100,HA-1004, HA-1077, HA-1100, hydroxyfasudil, indirubin-3′-oxime,5-Iodotubercidin, kenpaullone, KN-62, KY12420, LFM-A13, lavendustin A,luteolin, LY-294002, LY294002, mallotoxin, ML-9, NSC-154020, NSC-226080,NSC-231634, NSC-664704, NSC-680410, NU6102, olomoucine, oxindole I,PD-153035, PD-98059, PD 169316, phloretin, phloridzin, piceatannol,picropodophyllin, PK1, PP1, PP2, purvalanol A, quercetin, R406, R788,rapamune, rapamycin, Ro 31-8220, roscovitine, rottlerin, SB202190,SB203580, sirolimus, sorafenib, SL327, SP600125, staurosporine, STI-571,SU-11274, SU1498, SU4312, SU6656, 4,5,6,7-Tetrabromotriazole, TG101348,Triciribine, Tyrphostin AG 490, Tyrphostin AG 825, Tyrphostin AG 957,Tyrphostin AG 1024, Tyrphostin SU1498, U0126, VX-509, VX-667, VX-680,W-7, wortmannin, XL-019, XL-147, XL-184, XL-228, XL-281, XL-518, XL-647,XL-765, XL-820, XL-844, XL-880, Y-27632, ZD-1839, ZM-252868, ZM-447439,siRNA, miRNA Examples of phosphatase inhibitors include, but are notlimited to H₂O₂, siRNA, miRNA, Cantharidin, (−)-p-Bromotetramisole,Microcystin LR, Sodium Orthovanadate, Sodium Pervanadate, Vanadylsulfate, Sodium oxodiperoxo(1,10-phenanthroline)vanadate,bis(maltolato)oxovanadium(IV), Sodium Molybdate, Sodium Perm olybdate,Sodium Tartrate, Imidazole, Sodium Fluoride, β-Glycerophosphate, SodiumPyrophosphate Decahydrate, Calyculin A, Discodermia calyx, bpV(phen),mpV(pic), DMHV, Cypermethrin, Dephostatin, Okadaic Acid, NIPP-1,N-(9,10-Dioxo-9,10-dihydro-phenanthren-2-yl)-2,2-dimethyl-propionamide,α-Bromo-4-hydroxyacetophenone, 4-Hydroxyphenacyl Br,α-Bromo-4-methoxyacetophenone, 4-Methoxyphenacyl Br,α-Bromo-4-(carboxymethoxy)acetophenone, 4-(Carboxymethoxy)phenacyl Br,and bis(4-Trifluoromethylsulfonamidophenyl)-1,4-diisopropylbenzene,phenyarsine oxide, Pyrrolidine Dithiocarbamate, and Aluminum fluoride.In some embodiments, the phosphatase inhibitor is H₂O₂.

In some embodiments, the modulator is an activator or an inhibitor. Insome embodiments, the modulators are independently selected from thegroup consisting of growth factor, cytokine, adhesion moleculemodulator, hormone, small molecule, polynucleotide, antibodies, naturalcompounds, lactones, chemotherapeutic agents, immune modulator,carbohydrate, proteases, ions, reactive oxygen species, and radiation.In some embodiments, at least one modulator is a B cell receptormodulator. In some embodiments, the B cell receptor modulator is a Bcell receptor activator, such as Rituxan or a cross-linker of the B cellreceptor complex or the B-cell co-receptor complex.

In some embodiments, the modulator is PMA, BAFF, April, SDF1a, SCF,CD40L, IGF-1, Imiquimod, polyCpG, fludarabine, cyclophosphamide,chlorambucil IL-7, IL-6, IL-10, IL-27, IL-4, IL-2, IL-3, thapsigarginand/or a combination thereof.

In some embodiments, the activatable element is a protein. In someembodiments, the protein is selected from the group consisting of Akt1,Akt2, Akt3, SAPK/JNK1,2,3, p38s, Erk1/2, Syk, ZAP70, Btk, BLNK, Lck,PLCγ, PLC1γ₂, STAT1, STAT 3, STAT 4, STAT 5, STAT 6, CREB, Lyn, p-S6,Cbl, NF-κB, GSK3β, CARMA/Bcl10 and Tcl-1.

In another aspect, the invention provides methods of classifying a cellpopulation by contacting the cell population with at least onemodulator, where the modulator is F(ab)2 IgM, Rituxan, Alemtuzumab, antiCD22 (epratuzumab), anti-CD23 (lumiliximab), Campath, H₂O₂, PMA, BAFF,April, SDF1a, CD40L, IGF-1, Imiquimod, polyCpG, fludarabine,cyclophosphamide, chlorambucil, IL-7, IL-6, IL-10, IL-27, IL-4, IL-2,IL-3, thapsigargin and/or a combination thereof, determining thepresence or absence of an increase in activation level of an activatableelement in the cell population, and classifying the cell populationbased on the presence or absence of the increase in the activation ofthe activatable element.

In some embodiments the cell population is a hematopoietic-derived cellpopulation. In some embodiments, the hematopoietically derived cellpopulation is selected from the group consisting of pluripotenthematopoietic stem cells, B-lymphocyte lineage progenitor or derivedcells, T-lymphocyte lineage progenitor or derived cells, NK cell lineageprogenitor or derived cells, granulocyte lineage progenitor or derivedcells, monocyte lineage progenitor or derived cells, megakaryocytelineage progenitor or derived cells and erythroid lineage progenitor orderived cells. In some embodiments, the hematopoietic derived cellpopulation is a B-lymphocyte lineage progenitor and derived cellpopulation, e.g., an early pro-B cell population, late pro-B cellpopulation, large pre-B cell population, small pre-B cell population,immature B cell population, mature B cell population, plasma cell andmemory B cell population, a CD5+ B cell population, a CD38+ B cell, a Bcell bearing a mutatated or non mutated heavy chain of the B cellreceptor, or a B cell population expressing Zap70.

In some embodiments, the classification includes classifying the cellpopulation as a cell population that is correlated with a clinicaloutcome. In some embodiments, the clinical outcome is the predictedrespose to a specific therapy, or the prognosis and/or diagnosis of acondition. In some embodiments, the clinical outcome is the presence orabsence of a neoplastic or a hematopoietic condition, such asNon-Hodgkin Lymphoma, Hodgkin or other lymphomas, acute or chronicleukemias, polycythemias, thrombocythemias, multiple myeloma or plasmacell disorders, e.g., amyloidosis and Waldenstrom's macroglobulinemia,myelodysplastic disorders, myeloproliferative disorders, myelofibrosis,or atypical immune lymphoproliferations. In some embodiments, theneoplastic or hematopoietic condition is non-B lineage derived, such asacute myeloid leukemia (AML), Chronic Myeloid Leukemia (CML), non-B cellacute lymphocytic leukemia (ALL), non-B cell lymphomas, myelodysplasticdisorders, myeloproliferative disorders, myelofibrosis, polycythemias,thrombocythemias, or non-B atypical immune lymphoproliferations. In someembodiments, the neoplastic or hematopoietic condition is a B-Cell or Bcell lineage derived disorder, such as Chronic Lymphocytic Leukemia(CLL), B lymphocyte lineage leukemia, B lymphocyte lineage lymphoma,Multiple Myeloma, or plasma cell disorders, e.g., amyloidosis orWaldenstrom's macroglobulinemia. In some embodiments, the condition isCLL. In some embodiments, the CLL is defined by a monoclonal B cellpopulation that co-expresses CD5 with CD19 and CD23 or CD5 with CD20 andCD23 and dim surface immunoglobulin expression.

In some embodiments, the clinical outcome is the staging or grading of aneoplastic or hematopoietic condition. Examples of staging in methodsprovided by the invention include aggressive, indolent, benign,refractory, Roman Numeral staging, TNM Staging, Rai staging, Binetstaging, WHO classification, FAB classification, IPSS score, WPSS score,limited stage, extensive stage, staging according to cellular markerssuch as ZAP70, IgV_(H) mutation status and CD38, occult, includinginformation that may inform on time to progression, progression freesurvival, overall survival, or event-free survival.

In some embodiments of the methods of the invention, the classifying ofthe cell population based on activation level includes classifying thecell population as a cell population that is correlated to a patientresponse to a treatment, such as complete response, partial response,nodular partial response, no response, progressive disease, stabledisease, relapse or adverse reaction. The method may further comprisedetermining a method of treatment, e.g., chemotherapy, biologicaltherapy, targeted therapy, radiation therapy, bone marrowtransplantation, Peripheral stem cell transplantation, umbilical cordblood transplantation, autologous stem cell transplantation, allogeneicstem cell transplantation, syngeneic stem cell transplantation, surgery,induction therapy, maintenance therapy, watchful waiting, orholistic/alternative therapy.

In some embodiments of the methods of the invention, the classifying ofthe cell population based on activation level includes classifying thecell population as a cell population that is correlated with minimalresidual disease or emerging resistance.

BRIEF DESCRIPTION OF THE DRAWINGS

The novel features of the invention are set forth with particularity inthe appended claims. A better understanding of the features andadvantages of the present invention will be obtained by reference to thefollowing detailed description that sets forth illustrative embodiments,in which the principles of the invention are utilized, and theaccompanying drawings of which:

FIG. 1 depicts histograms showing the activation of p-Erk andp-Syk/pZap70 in Ramos cells following F(ab)₂IgM stimulation.

FIG. 2 depicts histograms showing the phosphorylation of BLNK, Cbl,PLCγ₂, Lck, and p38 in Ramos Cell lines following treatment withF(ab)₂IgM.

FIG. 3 depicts histograms showing increased phosphorylation of Erk inPheresed CLL Samples and in the Ramos cell lines following PMAactivation.

FIG. 4 depicts a histogram showing the activation of Erk in Pheresed CLLSamples and Ramos cells following activation with increasing amounts ofF(ab)₂IgM for 15 Min.

FIG. 5 depicts contour plots showing increased phosphorylation of Erkand Syk/Zap 70 in CLL Samples following treatment with PMA or F(ab)₂IgM.

FIG. 6 depicts contour plots showing phosphorylation of Erk andSyk/Zap70 in CLL samples following treatment with H₂O₂ alone or incombination with F(ab)₂IgM.

FIG. 7 depicts histograms showing the phosphorylation of Erk andSyk/Zap70 in healthy B cells following treatment with H₂O₂.

FIG. 8 depicts contour plots showing the phosphorylation of Erk andSyk/Zap70 in CLL cells upon treatment with H₂O₂ and F(ab)₂IgM.

FIG. 9 depicts contour plots showing Erk and Syk in CLL samplesfollowing activation with PMA or F(ab)₂IgM.

FIG. 10 depicts contour plots showing the phosphorylation of Erk andSyk/Zap70 following treatment with H₂O₂ alone or in combination withF(ab)₂IgM.

FIG. 11 depicts contour plots comparing CLL samples with low and highFrequency ZAP70 the phosphorylation or Erk and Syk/Zap70 followingtreatment with H₂O₂ alone or in combination with F(ab)₂IgM.

FIG. 12 depicts contour plots showing Syk/Zap70 and Erk phosphorylationafter treatment F(ab)₂IgM and H₂O₂ at different times in CLL samples.

FIG. 13 depicts contour plots showing Syk/Zap70 and Erk phosphorylationafter treatment with F(ab)₂IgM and H₂O₂ in CLL samples.

FIG. 14 depicts histograms showing the kinetics of signaling mediated byF(ab)₂IgM and H₂O₂ in CLL.

FIG. 15 depicts contour plots showing the influence of % ZAP70 on BLNKphosphorylation following treatment with H₂O₂ alone or in combinationwith F(ab)₂IgM.

FIG. 16 depicts histograms showing the influence of ZAP70 status on BLNKphosphorylation.

FIG. 17 depicts histograms showing the kinetics of phosphorylation ofpPLCy₂, prpS6, and pCbl after B-Cell Receptor crosslinking by F(ab)₂IgMAlone in CD20+/CD5+ population of CLL samples.

FIG. 18 depicts histograms showing the kinetics of phosphorylation ofpPLCy₂, prpS6, and pCbl after B-Cell Receptor crosslinking by F(ab)₂IgMand H₂O₂ in CD20+/CD5+ population in CLL samples.

FIG. 19 depicts histograms showing the kinetics of phosphorylation ofpPLCy₂, prpS6, and pCbl after B-Cell Receptor crosslinking by F(ab)₂IgMAlone in CD20+/CD5+ population in CLL samples.

FIG. 20 depicts histograms showing the kinetics of phosphorylation ofpPLCy₂, prpS6, and pCbl after B-Cell Receptor crosslinking by F(ab)₂IgMand H₂O₂ in CD20+/CD5+ population in CLL samples.

FIG. 21 depicts contour plots showing the phosphorylation of Syk/Zap 70and Erk by F(ab)₂IgM alone over time.

FIG. 22 depicts contour plots showing the phosphorylation of Syk/Zap 70and Erk in response to F(ab)₂IgM alone over time.

FIG. 23 depicts histograms showing phosphorylation of Syk, Erk and BLNKin response to F(ab)₂IgM over time in the CD20+/CD5+ population of CLLsamples.

FIG. 24 depicts histograms showing the kinetics of phosphorylation ofpPLCy₂, prpS6, and pCbl after H₂O₂ treatment in CD20+/CD5+ population ofCLL samples.

FIG. 25 depicts histograms showing the kinetics of phosphorylation ofpPLCy₂, prpS6, and pCbl after H₂O₂ treatment in CD20+/CD5+ population ofCLL Samples.

FIG. 26 depicts a heat-map showing intracellular responses of CLLpatient peripheral blood samples to BCR and/or H₂O₂ stimulation. CLLindicates that the sample was taken from a patient diagnosed with CLL.CON indicates that the sample was taken from a healthy subject. Thepatient sample numbers are indicated at the top of the heat map and eachcolumn represents a single patient. The shaded squares within the columnrepresent a phospho-protein node. The different shades represent thedegree of phosphorylation of each node (see scale at top of figure).Bright white (located to the far right of the scale) represents thegreatest increase (i.e. +3.0 Log Change). Black (located in the centerof the scale) indicates little or no change and dark grey (located tothe far left of the scale) represents the greatest decrease inphosphorylation status (i.e. −3.0 Log Change). These levels ofphosphorylation are derived from an equation which calculates thelog10-fold increase, or decrease, in median fluorescence intensity(MFI), of a stimulated sample divided by the MFI of an unstimulatedsample. The rows of the heatmap indicate the identity of thephospho-protein that was analyzed. For example, the label“p-Blnk/H₂O₂/F(ab)2 IgM” indicates that the phosphorylation status ofphosphorylated BLNK was measured in response to hydrogen peroxide (H₂O₂)and the Fab fragment that recognizes IgM (F(ab)2 IgM). “US” indicatesthat the sample was unstimulated.

FIG. 27 depicts the lower portion of the heat map shown in FIG. 26.White framed boxes show 2 distinct clusters of CLL patient samples inwhich signaling increases in response to H₂O₂ treatment (left hand side)or signaling decreases in response to H₂O₂ treatment (righthand side).

FIG. 28 depicts the lower portion of the heat map shown in FIG. 26further illustrating the two patient clusters, 10/22 patients (top leftwhite framed box) and 11/22 patients (bottom right white framed box),that are distinguished by H₂O₂ treatment. The cartoon on the right ofthe figure depicts the B cell receptor signaling pathway.

DETAILED DESCRIPTION OF THE INVENTION

The present invention incorporates information disclosed in otherapplications and texts. The following patent and other publications arehereby incorporated by reference in their entireties: Haskell et al,Cancer Treatment, 5^(th) Ed., W.B. Saunders and Co., 2001; Alberts etal., The Cell, 4^(th) Ed., Garland Science, 2002; Vogelstein andKinzler, The Genetic Basis of Human Cancer, 2d Ed., McGraw Hill, 2002;Michael, Biochemical Pathways, John Wiley and Sons, 1999; Weinberg, TheBiology of Cancer, 2007; Immunobiology, Janeway et al. 7^(th) Ed.,Garland, and Leroith and Bondy, Growth Factors and Cytokines in Healthand Disease, A Multi Volume Treatise, Volumes 1A and 1B, Growth Factors,1996. Patents and applications that are also incorporated by referenceinclude U.S. Pat. Nos. 7,381,535 and 7,393,656 and U.S. Ser. Nos. (USSN)10/193,462; 11/655,785; 11/655,789; 11/655,821; 11/338,957, 61/048,886;61/048,920; 61/048,657; 61/079,766; 61/155,362; 61/079,579; 61/079,537;61/079,551; 61/087,555 and 61/085,789. Some commercial reagents,protocols, software and instruments that are useful in some embodimentsof the present invention are available at the Becton Dickinson Websitehttp://www.bdbiosciences.com/features/products/, and the Beckman Coulterwebsite, http://www.beckmancoulter.com/Default.asp?bhfv=7. Relevantarticles include High-content single-cell drug screening withphosphospecific flow cytometry, Krutzik et al., Nature Chemical Biology,23 December (2007); Irish et al., FLt3 ligand Y591 duplication and Bcl-2over expression are detected in acute myeloid leukemia cells with highlevels of phosphorylated wild-type p53, Neoplasia, (2007), Irish et al.Mapping normal and cancer cell signaling networks: towards single-cellproteomics, Nature (2006) 6:146-155; and Irish et al., Single cellprofiling of potentiated phospho-protein networks in cancer cells, Cell,(2004) 118, 1-20; Schulz, K. R., et al., Single-cell phospho-proteinanalysis by flow cytometry, Curr Protoc Immunol, (2007) 78:8 8.17.1-20;Krutzik, P. O., et al., Coordinate analysis of murine immune cellsurface markers and intracellular phosphoproteins by flow cytometry, J.Immunol. (2005) 175(4):2357-65; Krutzik, P. O., et al., Characterizationof the murine immunological signaling network with phosphospecific flowcytometry, J. Immunol. (2005) 175(4):2366-73; Shulz et al., CurrentProtocols in Immunology (2007) 78:8.17.1-20; Stelzer et al. Use ofMultiparameter Flow Cytometry and Immunophenotyping for the Diagnosisand Classfication of Acute Myeloid Leukemia, Immunophenotyping, Wiley,2000; and Krutzik, P. O. and Nolan, G. P., Intracellular phospho-proteinstaining techniques for flow cytometry: monitoring single cell signalingevents, Cytometry A. (2003) 55(2):61-70; Hanahan D., Weinberg, TheHallmarks of Cancer, CELL (2000) 100:57-70; Krutzik et al, High contentsingle cell drug screening with phophosphospecific flow cytometry, NatChem. Biol. (2008) 4:132-42. Experimental and process protocols andother helpful information can be found athttp:/proteomices.stanford.edu. The articles and other references citedbelow are also incorporated by reference in their entireties for allpurposes.

Introduction

In some embodiments, this invention is directed to methods andcompositions for diagnosis, prognosis and to methods of treatment. Insome embodiments, the physiological status of cells present in a sample(e.g. clinical sample) is used, e.g., in diagnosis or prognosis of acondition, patient selection for therapy, to monitor treatment, modifytherapeutic regimens, and to further optimize the selection oftherapeutic agents; which may be administered as one or a combination ofagents. Hence, therapeutic regimens can be individualized and tailoredaccording to the data obtained prior to, and at different times over thecourse of treatment, thereby providing a regimen that is individuallyappropriate.

In some embodiments, the present invention is directed to methods forclassifying a sample derived from an individual having or suspected ofhaving a condition, e.g., a neoplastic or a hematopoietic condition. Theinvention allows for identification of prognostically andtherapeutically relevant subgroups of conditions and prediction of theclinical course of an individual. The methods of the invention providetools useful in the treatment of an individual afflicted with acondition, including but not limited to methods of choosing a therapyfor an individual, methods of predicting response to a therapy for anindividual, methods of determining the efficacy of a therapy in anindividual, methods for assigning a risk group, methods of predicting anincreased risk of relapse, methods of predicting an increased risk ofdeveloping secondary complications, and methods of determining theprognosis for an individual. The present invention provides methods thatcan serve as a prognostic indicator to predict the course of acondition, e.g. whether the course of a neoplastic or a hematopoieticcondition in an individual will be aggressive or indolent, therebyaiding the clinician in managing the patient and evaluating the modalityof treatment to be used.

In some embodiments, the invention is directed to methods fordetermining the activation level of one or more activatable elements ina cell upon treatment with one or more modulators. The activation of anactivatable element in the cell upon treatment with one or moremodulators can reveal operative pathways in a condition that can then beused, e.g., choose a therapy for an individual, predict response to atherapy for an individual, determine the efficacy of a therapy in anindividual. In some embodiments the modulators may themselves be useddirectly within individuals as therapeutic agents. In some embodimentsthe activation of an activateable agent may be used as an indicator topredict course of the condition, identify risk group, predict anincreased risk of developing secondary complications, and determine theprognosis for an individual.

In some embodiments, the invention is directed to methods forclassifying a cell by contacting the cell with an inhibitor, determiningthe presence or absence of an increase in activation level of anactivatable element in the cell, and classifying the cell based on thepresence or absence of the increase in the activation of the activatableelement. In some embodiments, the invention is directed to methods ofdetermining the presence or absence of a condition in an individual bysubjecting a cell from the individual to a modulator and an inhibitor,determining the activation level of an activatable element in the cell,and determining the presence or absence of the condition based on theactivation level upon treatment with a modulator and an inhibitor.

In some embodiments, the invention is directed to methods forclassifying a cell by contacting the cell with an inhibitor, determiningthe presence or absence of a change in activation level of anactivatable element in the cell, and classifying the cell based on thepresence or absence of the change in the activation of the activatableelement. In some embodiments the change is an increase. In someembodiments the change is a decrease.

In some embodiments, the invention is directed to methods of determiningtonic signaling status of a cell by subjecting the cell to a modulator,determining the activation level of an activatable element thatparticipates in a tonic signaling pathway in the cell, and determiningthe status of a tonic signaling pathway in the cell from the activationlevel. Tonic signaling in a cell may have functional consequences, forinstance, to maintain certain differentiated cellular properties orfunctions. In some embodiments of the invention, the status of a tonicsignaling pathway is used to correlate the status to differences inpopulations.

In some embodiments, the invention is directed to methods of determininga phenotypic profile of a population of cells by exposing the populationof cells in separate cultures to a plurality of modulators, wherein atleast one of the modulators is an inhibitor, determining the presence orabsence of an increase in activation level of an activatable element inthe cell population from each of the separate culture and classifyingthe cell population based on the presence or absence of the increase inthe activation of the activatable element from populations of cells ineach separate culture.

In some embodiments, the invention is directed to methods of classifyinga cell population by contacting the cell population with at least onemodulator, where the modulator is F(ab)2 IgM, H₂O₂, PMA, BAFF, April,SDFla, CD40L, IGF-1, Imiquimod, polyCpG, IL-7, IL-6, IL-10, IL-27, IL-4,IL-2, IL-3, thapsigargin and/or a combination thereof, determining thepresence or absence of an increase in activation level of an activatableelement in the cell population, and classifying the cell populationbased on the presence or absence of the increase in the activation ofthe activatable element.

In some embodiments, the invention is directed to methods of correlatingand/or classifying an activation state of a CLL cell with a clinicaloutcome in an individual by subjecting the CLL cell from the individualto a modulator, where the CLL cell expresses a B-Cell receptor (BCR),determining the activation levels of a plurality of activatableelements, and identifying a pattern of the activation levels of theplurality of activatable elements to determine the presence or absenceof an alteration in signaling proximal to the BCR, where the presence ofthe alteration is indicative of a clinical outcome.

In some embodiments a method for classifying a cell comprises contactingthe cell with an inhibitor, determining the presence or absence of achange in an activation level of at least one activatable element insaid cell, and classifying said cell based on said presence or absenceof said change in the activation level of said at least one activatableelement. In some embodiments the change is an increase. In someembodiments the change is a decrease.

In some embodiments the method of classifying a cell further comprisesdetermining the level of an intracellular marker, cell surface marker orany combination thereof. For example a cell may be classified by achange in activation level of an activatable element and also by thelevel of one or more cell surface markers. In addition a cell may beclassified by a change in activation level of an activatable element andby the level of an intracellular marker. Combinations may also be used.Serum markers are also useful in methods of diagnosis, prognosis,determining treatments effects and/or choosing a treatment.

One or more cell surface markers may also be used in the method of theinvention in addition to intracellular markers (e.g. phospho-proteins).In some embodiments, the method comprises determining the level of aplurality of cell surface markers. Cell surface markers may include anycell surface molecule that is detected by flow cytometry. In someembodiments the cell surface marker is a human leukocyte differentiationantigen. In some embodiments the human leukocyte differentiation antigenis selected from the list: CD1, CD2, CD3, CD4, CD5, CD8, CD10, CD14,CD19, CD20, CD22, CD23, CD40, CD52, CD100, CD280, CD281, CD282, CD283,CD284, and CD289. In some embodiments the human leukocytedifferentiation antigen is selected from the list comprising CD1 thoughCD300. In some embodiments the cell surface marker is any cell surfacereceptor or ligand. Examples of cell surface ligands and receptorsinclude, but are not limited to, members of the TNF superfamily,interleukins, hormones, neurotransmitters, interferons, growth factor,chemokines, integrins, toll receptor ligands, prostaglandins, orleukotriene families. Other examples of cell surface markers include,but are not limited to metalloproteases. In some embodiments the cellsurface marker is membrane bound IgM. In some embodiments the cellsurface marker is a B-cell receptor (BCR) or a component of a BCR. Insome embodiments the marker is CD45, CD5, CD14, CD19, CD20, CD22, CD23,CD27, CD37, CD40, CD52, CD79, CD38, CD96, major histocompatabilityantigen (MHC) Class1 or MHC Class 2. In some embodiments the cellsurface marker is membrane bound IgD. In some embodiments the cellsurface marker is membrane bound IgG. In some embodiments, the method ofclassifying a cell comprises determining a level of at least one cellsurface marker on said cell and an activation level of at least oneactivatable element on said cell. In some embodiments, the method ofclassifying a cell comprises determining the level of cell surface IgMon said cell. In some embodiments, the method comprises determining thelevel of cell surface IgD on said cell. In some embodiments, the methodcomprises determining the level of a BCR on said cell. In someembodiment the cell surface marker is associated with a diesease orconditions. In some embodiments the maker is CD38 or CD96. In someembodiments the marker is CD38 and the condition is leukemia. In someembodiments the marker is CD96 and the condition is leukemia.

One or more intracellular markers may be used in the method of theinvention. The levels of these markers can be determined before they aresecreted and are referred to as “captured”. Examples of capturedintracellular markers include, but are not limited to, TNF superfamilymembers, interleukins, hormones, neurotransmitters, interferons, growthfactors, chemokines, integrins, prostaglandins, leukotrines andreceptors for all of the above. Examples of intracellular markers alsoinclude, but are not limited to, metalloproteases. Examples ofintracellular markers also include, but are not limited to, proteinsinvolved in programmed cell death and proliferation. Examples ofintracellular markers also include, but are not limited to viruses,pathogens, parasites and components or products thereof. In someembodiments, the method of classifying a cell further comprisesdetermining the level of an intracellular pathogen or component of apathogen. In some embodiments the intracellular pathogen is HIV. In someembodiments the intracellular pathogen is EBV. In some embodiments theintracellular component of a pathogen is a nucleic acid sequence derivedfrom said pathogen. In some embodiments the intracellular component of apathogen is a pathogen derived polypeptide.

The method of the invention may comprise determining the level of one ormore serum markers. In some embodiments the serum marker is a marker ofa condition. In some embodiments the serum marker is a marker ofinflammation. In some embodiments the serum marker is a solublecytokine, TNF superfamily member, interleukin, hormone,neurotransmitter, interferon, growth factor, chemokine, integrin,prostaglandin, leukotriene or any soluble receptor thereof. In someembodiments the serum marker is a marker of a specific disease orcondition. In some embodiments the serum marker is a cancer marker. Insome embodiments the serum marker is a leukemia marker. In someembodiments the serum marker is beta-2-microglobulin, calcitonin, CD20,CD23, CD52, IL6, IL2R, ICAM-1, CD14, IgG, thymidine kinase or ferritin.In some embodiments the serum marker is a pharmaceutical drug, pathogen,virus, parasite, small compound or toxin. Therefore, in someembodiments, the methods described herein are for diagnosis, prognosisor determining a method of treatment for a subject or patient. In someembodiments the methods comprise classifying a cell or population ofcells. In certain embodiments, the methods of diagnosis, prognosis ordetermining a method of treatment comprise determining the level of atleast one serum marker derived from the subject or patient. In someembodiments the serum marker is a cytokine, chemokine, soluble receptor,growth factor, antibody or binding protein. In some embodiments theserum marker is a pathogen. In some embodiments the serum marker is apharmaceutical compound or drug.

The subject invention also provides kits for use in determining thephysiological status of cells in a sample, the kit comprising one ormore specific binding elements for signaling molecules, and mayadditionally comprise one or more therapeutic agents. The kit mayfurther comprise a software package for data analysis of thephysiological status, which may include reference profiles forcomparison with the test profile.

Methods

In some embodiments, the invention provides methods, including methodsto determine the physiological status of a cell, e.g., by determiningthe activation level of an activatable element upon contact with one ormore modulators. In some embodiments, the invention provides methods,including methods to classify a cell according to the status of anactivatable element in a cellular pathway. The information can be usedin prognosis and diagnosis, including susceptibility to disease(s),status of a diseased state and response to changes, in the environment,such as the passage of time, treatment with drugs or other modalities.The physiological status of the cells provided in a sample (e.g.clinical sample) may be classified according to the-activation ofcellular pathways of interest. The cells can also be classified as totheir ability to respond to therapeutic agents and treatments.

One or more cells, or samples containing one or more cells, can beisolated from body samples, such as, but not limited to, smears, sputum,biopsies, secretions, cerebrospinal fluid, bile, blood, lymph fluid,urine and feces, a lavage of a tissue or organ (e.g. lung) or tissuewhich has been removed from organs, such as breast, lung, intestine,skin, cervix, prostate, and stomach. For example, a tissue sample cancomprise a region of functionally related cells or adjacent cells. Suchsamples can comprise complex populations of cells, which can be assayedas a population, or separated into sub-populations. Such cellular andacellular samples can be separated by centrifugation, elutriation,density gradient separation, apheresis, affinity selection, panning,FACS, centrifugation with Hypaque, etc. By using antibodies specific formarkers identified with particular cell types, a relatively homogeneouspopulation of cells may be obtained. Alternatively, a heterogeneous cellpopulation can be used. Cells can also be separated by using filters.For example, whole blood can also be applied to filters that areengineered to contain pore sizes that select for the desired cell typeor class. Rare pathogenic cells can be filtered out of diluted, wholeblood following the lysis of red blood cells by using filters with poresizes between 5 to 10 μm, as disclosed in U.S. patent application Ser.No. 09/790,673. Other devices can separate tumor cells from thebloodstream, see Demirci U, Toner M., Direct etch method formicrofluidic channel and nanoheight post-fabrication by picoliterdroplets, Applied Physics Letters 2006; 88 (5), 053117; and Irimia D,Geba D, Toner M., Universal microfluidic gradient generator, AnalyticalChemistry 2006; 78: 3472-3477. Once a sample is obtained, it can be useddirectly, frozen, or maintained in appropriate culture medium for shortperiods of time. Methods to isolate one or more cells for use accordingto the methods of this invention are performed according to standardtechniques and protocols well-established in the art.

Suitable cells include those cell types associated in a wide variety ofdisease conditions, even while in a non-diseased state. Accordingly,suitable eukaryotic cell types include, but are not limited to, tumorcells of all types (e.g. melanoma, myeloid leukemia, carcinomas of thelung, breast, ovaries, colon, kidney, prostate, pancreas and testes),cardiomyocytes, dendritic cells, endothelial cells, epithelial cells,lymphocytes (T-cell and B cell), mast cells, eosinophils, vascularintimal cells, macrophages, natural killer cells, erythrocytes,hepatocytes, leukocytes including mononuclear leukocytes, stem cellssuch as haemopoetic, neural, skin, lung, kidney, liver and myocyte stemcells (for use in screening for differentiation and de-differentiationfactors), osteoclasts, chondrocytes and other connective tissue cells,keratinocytes, melanocytes, liver cells, kidney cells, and adipocytes.Suitable cells also include primary disease state cells, such as primarytumor cells. Suitable cells also include known research cells,including, but not limited to, Jurkat T cells, NIH3T3 cells, CHO, COS,etc. See the ATCC cell line catalog, hereby expressly incorporated byreference.

In some embodiments, the cells are cultured post collection in a mediasuitable for revealing the activation level of an activatable element(e.g. RPMI, DMEM) in the presence, or absence, of serum such as fetalbovine serum, bovine serum, human serum, porcine serum, horse serum, orgoat serum. When serum is present in the media it could be present at alevel ranging from 0.0001% to 100%. In some embodiments serum is presentin the media at a level ranging from 0.0001% to 90%. In some embodimentsserum is present in the media at a level ranging from 0.01% to 30%. Insome embodiments serum is present in the media at 1, 2, 3, 4, 5, 6, 7,8, 9 or 10%. In some embodiments, serum is present in the media at anysuitable level.

In some embodiments, the cell is a hematopoietic cell. Examples ofhematopoietic cells include but are not limited to pluripotenthematopoietic stem cells, B-lymphocyte lineage progenitor or derivedcells, T-lymphocyte lineage progenitor or derived cells, NK cell lineageprogenitor or derived cells, granulocyte lineage progenitor or derivedcells, monocyte lineage progenitor or derived cells, megakaryocytelineage progenitor or derived cells and erythroid lineage progenitor orderived cells.

In some embodiments, the cells used in the present invention are takenfrom a patient. Cells used in the present invention can be purified fromwhole blood by any suitable method.

The term “patient” or “individual” as used herein includes humans aswell as other mammals. The methods generally involve determining thestatus of an activatable element. The methods also involve determiningthe status of a plurality of activatable elements.

In some embodiments, the invention provides a method of classifying acell by determining the presence or absence of a change in activationlevel of an activatable element in the cell upon treatment with one ormore modulators, and classifying the cell based on the presence orabsence of the change in the activation of the activatable element. Insome embodiments the change is a decrease. In some embodiments thechange is an increase. In some embodiments of the invention, theactivation level of the activatable element is determined by contactingthe cell with a binding element that is specific for an activation stateof the activatable element. In some embodiments, a cell is classifiedaccording to the activation level of a plurality of activatable elementsafter the cell have been subjected to a modulator. In some embodimentsof the invention, the activation levels of a plurality of activatableelements are determined by contacting a cell with a plurality of bindingelement, where each binding element is specific for an activation stateof an activatable element.

The classification of a cell according to the status of an activatableelement can comprise classifying the cell as a cell that is correlatedwith a clinical outcome. In some embodiments, the clinical outcome isthe prognosis and/or diagnosis of a condition. In some embodiments, theclinical outcome is the presence or absence of a neoplastic or ahematopoietic condition such as Non-Hodgkin Lymphoma, Hodgkin or otherlymphomas, acute or chronic leukemias, polycythemias, thrombocythemias,multiple myeloma or plasma cell disorders, e.g., amyloidosis andWaldenstrom's macroglobulinemia, myelodysplastic disorders,myeloproliferative disorders, myelofibrosis, or atypical immunelymphoproliferations. In some embodiments, the neoplastic orhematopoietic condition is non-B lineage derived, such as Acute myeloidleukemia (AML), Chronic Myeloid Leukemia (CML), non-B cell Acutelymphocytic leukemia (ALL), non-B cell lymphomas, myelodysplasticdisorders, myeloproliferative disorders, myelofibrosis, polycythemias,thrombocythemias, or non-B atypical immune lymphoproliferations, ChronicLymphocytic Leukemia (CLL), B lymphocyte lineage leukemia, B lymphocytelineage lymphoma, Multiple Myeloma, or plasma cell disorders, e.g.,amyloidosis or Waldenstrom's macroglobulinemia. In some embodiments, theclinical outcome is the presence or absence of a neoplastic or ahematopoietic condition, such as Chronic Lymphocytic Leukemia (CLL), Blymphocyte lineage leukemia, B lymphocyte lineage lymphoma, MultipleMyeloma, acute lymphoblastic leukemia (ALL), B-cell pro-lymphocyticleukemia, precursor B lymphoblastic leukemia, hairy cell leukemia orplasma cell disorders, e.g., amyloidosis or Waldenstrom'smacroglobulinemia, B cell lymphomas including but not limited to diffuselarge B cell lymphoma, follicular lymphoma, mucosa associated lymphatictissue lymphoma, small cell lymphocytic lymphoma, mantle cell lymphomaand marginal zone lymphoma. In some embodiments, the condition is CLL.In some embodiments, the clinical outcome is the staging or grading of aneoplastic or hematopoietic condition. Examples of staging include, butare not limited to, aggressive, indolent, benign, refractory, RomanNumeral staging, TNM Staging, Rai staging, Binet staging, WHOclassification, FAB classification, IPSS score, WPSS score, limitedstage, extensive stage, staging according to cellular markers such asZAP70 and CD38, occult, including information that may inform on time toprogression, progression free survival, overall survival, or event-freesurvival.

The classification of a cell according to the status of an activatableelement can comprise classifying a cell as a cell that is correlated toa patient response to a treatment. In some embodiments, the patientresponse is selected from the group consisting of complete response,partial response, nodular partial response, no response, progressivedisease, stable disease and adverse reaction.

The classification of a cell according to the status of an activatableelement can comprise classifying the cell as a cell that is correlatedwith minimal residual disease or emerging resistance.

The classification of a cell according to the status of an activatableelement can comprise selecting a method of treatment. Example of methodsof treatments include, but are not limited to, chemotherapy, biologicaltherapy, radiation therapy, bone marrow transplantation, Peripheral stemcell transplantation, umbilical cord blood transplantation, autologousstem cell transplantation, allogeneic stem cell transplantation,syngeneic stem cell transplantation, surgery, induction therapy,maintenance therapy, watchful waiting, and holistic/alternative therapy.

Modulators include compounds or conditions capable of impacting cellularsignaling networks. A modulator can be an activator or an inhibitor.Modulators can take the form of a wide variety of environmental inputs.Examples of modulators include but are not limited to growth factors,cytokines, chemokines, soluble receptors, Toll-like receptor ligands,pathogens, parasites, components of pathogens or parasites, adhesionmolecule modulators, pharmaceutical compounds, drugs, hormones, smallmolecules, polynucleotides, antibodies, natural compounds, lactones,chemotherapeutic agents, immune modulators, carbohydrates, proteases,ions, reactive oxygen species, radiation, physical parameters such asheat, cold, UV radiation, peptides, and protein fragments, either aloneor in the context of cells, cells themselves, viruses, and biologicaland non-biological complexes (e.g. beads, plates, viral envelopes,antigen presentation molecules such as major histocompatibilitycomplex). Examples of modulators include, but are not limited to, F(ab)2IgM, Rituxan, alemtuzumab, fludarabine, cyclophosphamide, chlorambucil,anti CD22 (epratuzumab), anti CD23 (lumiliximab), H₂O₂, PMA, BAFF,April, SDF1a, CD40L, IGF-1, Imiquimod, polyCpG, IL-7, IL-6, IL-10,IL-27, IL-4, IL-2, and IL-3. Additional modulators, inhibitors andactivators are disclosed in U.S. 61/085,789 which is hereby incorporatedby reference in its entirety.

In some embodiments, the modulator is an activator. In some embodimentsthe modulator is an inhibitor. In some embodiments, the inventionprovides methods for classifying a cell by contacting the cell with aninhibitor, determining the presence or absence of a change in activationlevel of an activatable element in the cell, and classifying the cellbased on the presence or absence of the change in the activation of theactivatable element. In some embodiments the change is a decrease. Insome embodiments the change is an increase. In some embodiments, a cellis classified according to the activation level of a plurality ofactivatable elements after the cell have been subjected to an inhibitor.In some embodiments, the inhibitor is an inhibitor of a cellular factoror a plurality of factors that participates in a signaling cascade inthe cell. In some embodiments, the inhibitor is a kinase or phosphataseinhibitor. Examples of kinase inhibitors include adaphostin, AG 490, AG825, AG 957, AG 1024, aloisine, aloisine A, alsterpaullone,aminogenistein, API-2, apigenin, arctigenin, AY-22989, BAY 61-3606,bisindolylmaleimide IX, chelerythrine,10-[4′-(N,N-Diethylamino)butyl]-2-chlorophenoxazine hydrochloride,dasatinib, 2-Dimethylamino-4,5,6,7-tetrabromo-1H-benzimidazole,5,7-Dimethoxy-3-(4-pyridinyl)quinoline dihydrochloride, edelfosine,ellagic acid, enzastaurin, ER 27319 maleate, erlotinib, ET18OCH3,fasudil, flavopiridol, gefitinib, GW 5074, H-7, H-8, H-89, HA-100,HA-1004, HA-1077, HA-1100, hydroxyfasudil, indirubin-3′-oxime,5-Iodotubercidin, kenpaullone, KN-62, KY12420, LFM-A13, lavendustin A,luteolin, LY-294002, LY294002, mallotoxin, ML-9, NSC-154020, NSC-226080,NSC-231634, NSC-664704, NSC-680410, NU6102, olomoucine, oxindole I,PD-153035, PD-98059, PD 169316, phloretin, phloridzin, piceatannol,picropodophyllin, PK1, PP1, PP2, purvalanol A, quercetin, R406, R788,rapamune, rapamycin, Ro 31-8220, roscovitine, rottlerin, SB202190,SB203580, sirolimus, sorafenib, SL327, SP600125, staurosporine, STI-571,SU-11274, SU1498, SU4312, SU6656, 4,5,6,7-Tetrabromotriazole, TG101348,Triciribine, Tyrphostin AG 490, Tyrphostin AG 825, Tyrphostin AG 957,Tyrphostin AG 1024, Tyrphostin SU1498, U0126, VX-509, VX-667, VX-680,W-7, wortmannin, XL-019, XL-147, XL-184, XL-228, XL-281, XL-518, XL-647,XL-765, XL-820, XL-844, XL-880, Y-27632, ZD-1839, ZM-252868, ZM-447439,Examples of phosphatase inhibitors include, but are not limited to H₂O₂,siRNA, miRNA, Cantharidin, (−)-p-Bromotetramisole, Microcystin LR,Sodium Orthovanadate, Sodium Pervanadate, Vanadyl sulfate, Sodiumoxodiperoxo(1,10-phenanthroline)vanadate, bis(maltolato)oxovanadium(IV),Sodium Molybdate, Sodium Perm olybdate, Sodium Tartrate, Imidazole,Sodium Fluoride, β-Glycerophosphate, Sodium Pyrophosphate Decahydrate,Calyculin A, Discodermia calyx, bpV(phen), mpV(pic), DMHV, Cypermethrin,Dephostatin, Okadaic Acid, NIPP-1,N-(9,10-Dioxo-9,10-dihydro-phenanthren-2-yl)-2,2-dimethyl-propionamide,α-Bromo-4-hydroxyacetophenone, 4-Hydroxyphenacyl Br,α-Bromo-4-methoxyacetophenone, 4-Methoxyphenacyl Br,α-Bromo-4-(carboxymethoxy)acetophenone, 4-(Carboxymethoxy)phenacyl Br,and bis(4-Trifluoromethylsulfonamidophenyl)-1,4-diisopropylbenzene,phenyarsine oxide, Pyrrolidine Dithiocarbamate, and Aluminum fluoride.In some embodiments, the phosphatase inhibitor is H₂O₂.

In some embodiments, the methods of the invention provide methods fordetermining the presence or absence of a condition in an individual bysubjecting a cell from the individual to a modulator and an inhibitor,determining the activation level of an activatable element in the cell,and determining the presence or absence of a condition based on theactivation level. In some embodiments, the activation level of aplurality of activatable elements in the cell is determined. Theinhibitor can be an inhibitor as described herein. In some embodiments,the inhibitor is a phosphatase inhibitor. In some embodiments, theinhibitor is H₂O₂. The modulator can be any modulator described herein.In some embodiments, the modulator is a B cell receptor modulator. Insome embodiments, the B cell receptor modulator is a B cell receptoractivator. An example of B cell receptor activator is a cross-linker ofthe B cell receptor complex or the B-cell co-receptor complex. In someembodiments, cross-linker is an antibody or molecular binding entity. Insome embodiments, the cross-linker is an antibody. In some embodiments,the antibody is a multivalent antibody. In some embodiments, theantibody is a monovalent, bivalent, or multivalent antibody made moremultivalent by attachment to a solid surface or tethered on ananoparticle surface to increase the local valency of the epitopebinding domain.

The cross-linker can be a molecular binding entity. In some embodiments,the molecular binding entity acts upon or binds the B cell receptorcomplex via carbohydrates or an epitope in the complex. In someembodiments, the molecular is a monovalent, bivalent, or multivalent ismade more multivalent by attachment to a solid surface or tethered on ananoparticle surface to increase the local valency of the epitopebinding domain.

The cross-linking of the B cell receptor complex or the B-cellco-receptor complex can comprise binding of an antibody or molecularbinding entity to the cell and then causing its crosslinking viainteraction of the cell with a solid surface that causes crosslinking ofthe BCR complex via antibody or molecular binding entity.

The crosslinker can be F(ab)2 IgM, IgG, IgD, polyclonal BCR antibodies,monoclonal BCR antibodies, Fc receptor derived binding elements and/or acombination thereof. The Ig can be derived from a species selected fromthe group consisting of mouse, goat, rabbit, pig, rat, horse, cow,shark, chicken, llama or human. The Ig or binding element can be fullyhuman or partially human and can be generated by any suitable methodknown in the art. In some embodiments, the crosslinker is F(ab)2 IgM,Polyclonal IgM antibodies, Monoclonal IgM antibodies, BiotinylatedF(ab)2 IgCM, Biotinylated Polyclonal IgM antibodies, BiotinylatedMonoclonal IgM antibodies and/or a combination thereof.

In some embodiments, the methods of the invention provides for the useof more than one modulator. In some embodiments, the methods of theinvention utilize a B cell receptor activator and a phosphataseinhibitor. In some embodiments, the methods of the invention utilizeF(ab)2IgM or biotinylated F(ab)2IgM and H₂O₂.

In some embodiments, the methods of the invention provides for methodsof classifying a cell population by exposing the cell population inseparate cultures to a plurality of modulators and determining thestatus of activatable elements in the cell populations. In someembodiments, the status of a plurality of activatable elements in thecell population is determined. In some embodiments, at least one of themodulators of the plurality of modulators is an inhibitor. The modulatorcan be any modulators described herein. In some embodiments, themodulator is selected from the group consisting of F(ab)2 IgM, H₂O₂,PMA, BAFF, April, SDFla, CD40L, IGF-1, Imiquimod, polyCpG, IL-7, IL-6,IL-10, IL-27, IL-4, IL-2, IL-3, thapsigargin and a combination thereof.In some embodiments of the invention, the status of an activatableelement is determined by contacting the cell population with a bindingelement that is specific for an activation state of the activatableelement. In some embodiments, the status of a plurality of activatableelements is determined by contacting the cell population with aplurality of binding elements, where each binding element is specificfor an activation state of an activatable element.

In some embodiments, the methods of the invention provide for methodsfor classifying a cell population by contacting the cell population withat least one modulator, where the modulator is to F(ab)2 IgM, Rituxan,Alemtuzumab, anti CD22 (epratuzumab), anti-CD23 (lumiliximab), Campath,H₂O₂, PMA, BAFF, April, SDF1a, CD40L, IGF-1, Imiquimod, polyCpG,fludarabine, cyclophosphamide, chlorambucil, IL-7, IL-6, IL-10, IL-27,IL-4, IL-2, IL-3, thapsigargin and/or a combination thereof, anddetermining the status of an activatable element in the cell population.In some embodiments, the status of a plurality of activatable elementsin the cell population is determined. In some embodiments of theinvention, the status of an activatable element is determined bycontacting the cell population with a binding element that is specificfor an activation state of the activatable element. In some embodiments,the status of a plurality of activatable elements is determined bycontacting the cell population with a plurality of binding elements,where each binding element is specific for an activation state of anactivatable element.

In some embodiments, the methods of the invention provide fordetermining a phenotypic profile of a population of cells by exposingthe population of cells in separate cultures to a plurality ofmodulators, wherein at least one of the modulators is an inhibitor,determining the presence or absence of a change in activation level ofan activatable element in the cell population from each of the separatecultures and classifying the cell population based on the presence orabsence of the change in the activation of the activatable element fromeach of the separate cultures. In some embodiments the change is adecrease. In some embodiments the change is an increase. In someembodiments, the modulator is selected from the group consisting ofF(ab)2 IgM, Rituxan, Alemtuzumab, anti CD22 (epratuzumab), anti-CD23(lumiliximab), Campath, H₂O₂, PMA, BAFF, April, SDFla, CD40L, IGF-1,Imiquimod, polyCpG, fludarabine, cyclophosphamide, chlorambucil, IL-7,IL-6, IL-10, IL-27, IL-4, IL-2, IL-3, thapsigargin and combinationthereof. In some embodiments, the status of a plurality of activatableelements in the cell population is determined. In some embodiments, thephenotypic profile of a population of cells is used to classify thepopulation as described herein. In some embodiments, the presence orabsence of an increase in the activation level of an activatable elementis determined by contacting the cell population with a binding elementthat is specific for an activation state of the activatable element. Insome embodiments, the status of a plurality of activatable elements isdetermined by contacting the cell population with a plurality of bindingelements, where each binding element is specific for an activation stateof an activatable element.

In some embodiments, the invention provides a method for classifying aB-lymphocyte progenitor or derived cell as described herein bycontacting the cell with a modulator, determining the presence orabsence of a change in activation level of an activatable element in thecell, and classifying the cell based on the presence or absence of thechange in the activation of the activatable element. In some embodimentsthe change is a decrease. In some embodiments the change is an increase.In some embodiments, the presence or absence of a change in theactivation level of an activatable element is determined by contactingthe cell with a binding element that is specific for an activation stateof the activatable element. In some embodiments, a B-lymphocyteprogenitor or derived cell is classified according to the activationlevel of a plurality of activatable elements after the cells have beensubjected to a modulator. In some embodiments, the presence or absenceof a change in the activation levels of a plurality of activatableelements is determined by contacting the cell population with aplurality of binding elements, where each binding elements is specificfor an activation state of an activatable element. In some embodiments,the method for classifying a B-lymphocyte progenitor or derived cellfurther comprises determining the level of at least one cell-surfacemarker. In some embodiments, the method for classifying a B-lymphocyteprogenitor or derived cell further comprises determining the level of atleast one intracellular marker, for example a captured intracellularcytokine. In some embodiments, the B-lymphocyte progenitor or derivedcell is associated with a condition such a neoplastic or hematopoeticcondition. Thus, in some embodiments, the invention provides methods forclassifying a B-lymphocyte progenitor or derived cell associated with acondition (e.g. neoplastic or hematopoetic condition) by contacting thecell with a modulator, determining the presence or absence of a changein activation level of one or more activatable elements in the cell, andclassifying the cell based on the presence or absence of the change inthe activation of the one or more activatable elements. In someembodiments the change is a decrease. In some embodiments the change isan increase.

In some embodiments, the invention provides methods for correlatingand/or classifying an activation state of a CLL cell with a clinicaloutcome in an individual by subjecting the CLL cell from the individualto a modulator, wherein the CLL cell expresses B-Cell receptor (BCR),determining the activation levels of a plurality of activatableelements, and identifying a pattern of the activation levels of theplurality of activatable elements to determine the presence or absenceof an alteration in signaling proximal to the BCR, wherein the presenceof the alteration is indicative of a clinical outcome. In someembodiments, the activation levels of a plurality of activatableelements are determined by contacting the cell with a plurality ofbinding elements, where each binding element is specific for anactivation state of an activatable element. The clinical outcome can beany clinical outcome described herein.

In some embodiments, the methods of the invention provide methods fordetermining tonic signaling status of a cell by subjecting the cell to amodulator, determining the activation level of an activatable elementthat participates in a tonic signaling pathway in the cell, anddetermining the status of a tonic signaling pathway in the cell from theactivation level. In some embodiments, the status of a plurality ofactivatable elements in the cell population is determined. In someembodiments, the activation level of an activatable element isdetermined by contacting the cell with a binding element that isspecific for an activation state of the activatable element. In someembodiments, the activation level of a plurality of activatable elementsis determined by contacting the cell with a plurality of bindingelements, where each binding element is specific for an activation stateof an activatable element. In some embodiments, the tonic signaling is acellular receptor tonic signaling. In some embodiments, the tonicsignaling is a T-cell receptor (TCR) tonic signaling. In someembodiments, the tonic signaling is a BCR tonic signaling. In someembodiments, the tonic signaling status in the cell is used to classifythe cell as described herein.

Patterns and profiles of one or more activatable elements are detectedusing the methods known in the art including those described herein. Insome embodiments, patterns and profiles of activatable elements that arecellular components of a cellular pathway are detected using the methodsdescribed herein. In some embodiments, patterns and profiles ofactivatable elements that are cellular components of a signaling pathwayare detected using the methods described herein. In some embodiments,patterns and profiles of activatable elements that are cellularcomponents of a tonic signaling pathway are detected using the methodsdescribed herein. For example, patterns and profiles of one or morephosphorylated polypeptide are detected using methods known in artincluding those described herein.

In some embodiments of the methods described herein, cells (e.g. normalnon-transformed cells) other than the cells associated with a condition(e.g. cancer cells) can be used to make clinical decisions. That is thatcells, other than cells associated with a condition (e.g. cancer cells),are in fact reflective of the condition process. Normal cells (e.g.healthy cells or non-transformed cells) can be used, e.g., in assigninga risk group, predicting an increased risk of relapse, predicting anincreased risk of developing secondary complications, choosing a therapyfor an individual, predicting response to a therapy for an individual,determining the efficacy of a therapy in an individual, and/ordetermining the prognosis for an individual. That is that cells otherthan cells associated with a condition (e.g. cancer cells) are in factreflective of the condition process. For instance, in the case ofcancer, infiltrating immune cells can determine the outcome of thedisease. In another aspect, a combination of information from a cancercell plus responding immune cells in the blood of a cancer patient canbe used for diagnosis or prognosis of the cancer.

Conditions

The methods of the invention are applicable to any condition in anindividual involving, indicated by, and/or arising from, in whole or inpart, altered physiological status in a cell. The term “physiologicalstatus” includes mechanical, physical, and biochemical functions in acell. In some embodiments, the physiological status of a cell isdetermined by measuring characteristics of cellular components of acellular pathway. Cellular pathways are well known in the art. In someembodiments the cellular pathway is a signaling pathway. Signalingpathways are also well known in the art (see, e.g., Hunter T., Cell(2000) 100(1): 113-27; Cell Signaling Technology, Inc., 2002 Catalogue,Pathway Diagrams pgs. 232-253). A condition involving or characterizedby altered physiological status may be readily identified, for example,by determining the state in a cell of one or more activatable elements,as taught herein.

In certain embodiments of the invention, the condition is a neoplasticor hematopoietic condition. In some embodiments, the neoplastic orhematopoietic condition is selected from the group consisting ofNon-Hodgkin Lymphoma, Hodgkin or other lymphomas, acute or chronicleukemias, polycythemias, thrombocythemias, multiple myeloma and plasmacell disorders, including amyloidosis and Waldenstrom'smacroglobulinemia, myelodysplastic disorders, myeloproliferativedisorders, myelofibrosis, and atypical immune lymphoproliferations.

In some embodiments, the neoplastic or hematopoietic condition is non-Blineage derived. Examples of non-B lineage derived neoplastic orhematopoietic condition include, but are not limited to, Acute myeloidleukemia (AML), Chronic Myeloid Leukemia (CML), non-B cell Acutelymphocytic leukemia (ALL), non-B cell lymphomas, myelodysplasticdisorders, myeloproliferative disorders, myelofibrosis, polycythemias,thrombocythemias, and non-B atypical immune lymphoproliferations.

In some embodiments, the neoplastic or hematopoietic condition is aB-Cell or B cell lineage derived disorder. Examples of B-Cell or B celllineage derived neoplastic or hematopoietic condition include but arenot limited to Chronic Lymphocytic Leukemia (CLL), B lymphocyte lineageleukemia, B lymphocyte lineage lymphoma, Multiple Myeloma, and plasmacell disorders, including amyloidosis and Waldenstrom'smacroglobulinemia.

In some embodiments, the condition is CLL. In some embodiments, CLL isdefined by a monoclonal B cell population that co-expresses CD5 withCD19 and CD23 or CD5 with CD20 and CD23 and by surface immunoglobulinexpression. In some embodiments, CLL is defined by a monoclonal B cellpopulation that co-expresses CD5 with CD19 and CD23 or CD5 with CD20 andCD23 and dim surface immunoglobulin expression.

Other conditions within the scope of the present invention include, butare not limited to, cancers such as gliomas, lung cancer, colon cancerand prostate cancer. Specific signaling pathway alterations have beendescribed for many cancers, including loss of PTEN and resultingactivation of Akt signaling in prostate cancer (Whang Y E. Proc NatlAcad Sci USA Apr. 28, 1998;95(9):5246-50), increased IGF-1 expression inprostate cancer (Schaefer et al., Science Oct. 9, 1998, 282: 199a), EGFRover expression and resulting ERK activation in glioma cancer (Thomas CY. Int J Cancer Mar. 10, 2003;104(1):19-27), expression of HER2 inbreast cancers (Menard et al. Oncogene. Sep. 29 2003, 22(42):6570-8),and APC mutation and activated Wnt signaling in colon cancer (Bienz M.Curr Opin Genet Dev 1999 October, 9(5):595-603).

Diseases other than cancer involving altered physiological status arealso encompassed by the present invention. For example, it has beenshown that diabetes involves underlying signaling changes, namelyresistance to insulin and failure to activate downstream signalingthrough IRS (Burks D J, White M F. Diabetes 2001 February; 50 Suppl1:S140-5). Similarly, cardiovascular disease has been shown to involvehypertrophy of the cardiac cells involving multiple pathways such as thePKC family (Malhotra A. Mol Cell Biochem 2001 September; 225(1-):97-107). Inflammatory diseases, such as rheumatoid arthritis, areknown to involve the chemokine receptors and disrupted downstreamsignaling (D'Ambrosio D. J Immunol Methods 2003 February; 273(1-2):3-13). The invention is not limited to diseases presently known toinvolve altered cellular function, but includes diseases subsequentlyshown to involve physiological alterations or anomalies.

In some embodiments, the present invention is directed to methods forclassifying one or more cells in a sample derived from an individualhaving or suspected of having condition. In some embodiments, theinvention allows for identification of prognostically andtherapeutically relevant subgroups of the conditions and prediction ofthe clinical course of an individual. In some embodiments, the inventionprovides method of classifying a cell according to the activation levelof one or more activatable element in a cell from an individual havingor suspected of having condition. In some embodiments, theclassification includes classifying the cell as a cell that iscorrelated with a clinical outcome. The clinical outcome can be theprognosis and/or diagnosis of a condition, and/or staging or grading ofa condition. In some embodiments, the classifying of the cell includesclassifying the cell as a cell that is correlated to a patient responseto a treatment. In some embodiments, the classifying of the cellincludes classifying the cell as a cell that is correlated with minimalresidual disease or emerging resistance.

Activatable Elements

The methods and compositions of the invention may be employed to examineand profile the status of any activatable element in a cellular pathway,or collections of such activatable elements. Single or multiple distinctpathways may be profiled (sequentially or simultaneously), or subsets ofactivatable elements within a single pathway or across multiple pathwaysmay be examined (again, sequentially or simultaneously).

As will be appreciated by those in the art, a wide variety of activationevents can find use in the present invention. In general, the basicrequirement is that the activation results in a change in theactivatable protein that is detectable by some indication (termed an“activation state indicator”), preferably by altered binding of alabeled binding element or by changes in detectable biologicalactivities (e.g., the activated state has an enzymatic activity whichcan be measured and compared to a lack of activity in the non-activatedstate). What is important is to differentiate, using detectable eventsor moieties, between two or more activation states (e.g. “off” and“on”).

The activation state of an individual activatable element is either inthe on or off state. As an illustrative example, and without intendingto be limited to any theory, an individual phosphorylatable site on aprotein can activate or deactivate the protein. The terms “on” and“off,” when applied to an activatable element that is a part of acellular constituent, are used here to describe the state of theactivatable element, and not the overall state of the cellularconstituent of which it is a part. Typically, a cell possesses aplurality of a particular protein or other constituent with a particularactivatable element and this plurality of proteins or constituentsusually has some proteins or constituents whose individual activatableelement is in the on state and other proteins or constituents whoseindividual activatable element is in the off state. Since the activationstate of each activatable element is measured through the use of abinding element that recognizes a specific activation state, only thoseactivatable elements in the specific activation state recognized by thebinding element, representing some fraction of the total number ofactivatable elements, will be bound by the binding element to generate ameasurable signal. The measurable signal corresponding to the summationof individual activatable elements of a particular type that areactivated in a single cell is the “activation level” for thatactivatable element in that cell.

Activation levels for a particular activatable element may vary amongindividual cells so that when a plurality of cells is analyzed, theactivation levels follow a distribution. The distribution may be anormal distribution, also known as a Gaussian distribution, or it may beof another type. Different populations of cells may have differentdistributions of activation levels that can then serve to distinguishbetween the populations. In some embodiments, the basis for classifyingcells is that the distribution of activation levels for one or morespecific activatable elements will differ among different phenotypes. Acertain activation level, or more typically a range of activation levelsfor one or more activatable elements seen in a cell or a population ofcells, is indicative that that cell or population of cells belongs to adistinctive phenotype. Other measurements, such as cellular levels(e.g., expression levels) of biomolecules that may not containactivatable elements, may also be used to classify cells in addition toactivation levels of activatable elements; it will be appreciated thatthese levels also will follow a distribution, similar to activatableelements. Thus, the activation level or levels of one or moreactivatable elements, optionally in conjunction with levels of one ormore levels of biomolecules that may not contain activatable elements,of cell or a population of cells may be used to classify a cell or apopulation of cells into a class. Once the activation level ofintracellular activatable elements of individual single cells is knownthey can be placed into one or more classes, e.g., a class thatcorresponds to a phenotype. A class encompasses a class of cells whereinevery cell has the same or substantially the same known activationlevel, or range of activation levels, of one or more intracellularactivatable elements. For example, if the activation levels of fiveintracellular activatable elements are analyzed, predefined classes thatencompass one or more of the intracellular activatable elements can beconstructed based on the activation level, or ranges of the activationlevels, of each of these five elements. It is understood that activationlevels can exist as a distribution and that an activation level of aparticular element used to classify a cell may be a particular point onthe distribution but more typically may be a portion of thedistribution.

In addition to activation levels of intracellular activatable elements,expression levels of intracellular or extracellular biomolecues, e.g.,proteins can be used alone or in combination with activation states ofactivatable elements to classify cells. Further, additional cellularelements, e.g., biomolecules or molecular complexes such as RNA, DNA,carbohydrates, metabolites, and the like, may be used in conjunctionwith activatable states or expression levels in the classification ofcells encompassed here.

In some embodiments, other characteristics that affect the status of acellular constituent may also be used to classify a cell. Examplesinclude the translocation of biomolecules or changes in their turnoverrates and the formation and disassociation of complexes of biomolecule.Such complexes can include multi-protein complexes, multi-lipidcomplexes, homo- or hetero-dimers or oligomers, and combinationsthereof. Other characteristics include proteolytic cleavage, e.g. fromexposure of a cell to an extracellular protease or from theintracellular proteolytic cleavage of a biomolecule.

Additional elements may also be used to classify a cell, such as theexpression level of extracellular or intracellular markers, nuclearantigens, enzymatic activity, protein expression and localization, cellcycle analysis, chromosomal analysis, cell volume, and morphologicalcharacteristics like granularity and size of nucleus or otherdistinguishing characteristics. For example, B cells can be furthersubdivided based on the expression of cell surface markers such as CD45,CD5, CD19, CD20, CD22, CD23, CD27, CD37, CD40, CD52, CD79, CD38, CD96,major histocompatability antigen (MHC) Class 1 or MHC Class 2.

Alternatively, predefined classes of cells can be classified based uponshared characteristics that may include inclusion in one or moreadditional predefined class or the presence of extracellular and/orintracellular markers, a similar gene expression profile, mutationalstatus, epigenetic silencing, nuclear antigens, enzymatic activity,protein expression and localization, cell cycle analysis, chromosomalanalysis, cell volume, and morphological characteristics likegranularity and size of nucleus or other distinguishing characteristics.

In some embodiments, the physiological status of one or more cells isdetermined by examining and profiling the activation level of one ormore activatable elements in a cellular pathway. In some embodiments, acell is classified according to the activation level of a plurality ofactivatable elements. In some embodiments, a hematopoietic cell isclassified according to the activation levels of a plurality ofactivatable elements. In some embodiments, the activation level of oneor more activatable elements of a hematopoietic cell is correlated witha condition. In some embodiments, the activation level of one or moreactivatable elements of a hematopoietic cell is correlated with aneoplastic or hematopoietic condition as described herein. Examples ofhematopoietic cells include but are not limited to pluripotenthematopoietic stem cells, myeloid progenitors, B-lymphocyte lineageprogenitor or derived cells, T-lymphocyte lineage progenitor or derivedcells, NK cell lineage progenitor or derived cells, granulocyte lineageprogenitor or derived cells, monocyte lineage progenitor or derivedcells, megakaryocyte lineage progenitor or derived cells and erythroidlineage progenitor or derived cells. In some embodiments, thehematopoietic cell is a B-lymphocyte lineage progenitor or derived cellas described herein.

In some embodiments, the activation level of one or more activatableelements in single cells within the sample is determined. Cellularconstituents that may include activatable elements include withoutlimitation, proteins, carbohydrates, lipids, nucleic acids andmetabolites. The activatable element may be a portion of the cellularconstituent, for example, an amino acid residue in a protein that mayundergo phosphorylation, or it may be the cellular constituent itself,for example, a protein that is activated by translocation from one partof the cell to another, change in conformation (due to, e.g., change inpH or ion concentration), by proteolytic cleavage, and the like. Uponactivation, a change occurs to the activatable element, such as covalentmodification of the activatable element (e.g., binding of a molecule orgroup to the activatable element, including but not limited to,phosphorylation, acetylation, methylation, ubiquitination) or aconformational change. Such changes generally contribute to changes inparticular biological, biochemical, or physical properties of thecellular constituent that contains the activatable element. The state ofthe cellular constituent that contains the activatable element isdetermined to some degree, though not necessarily completely, by thestate of activation of a particular activatable element of the cellularconstituent. For example, a protein may have multiple activatableelements, and the particular activation states of these elements mayoverall determine the activation state of the protein; the state of asingle activatable element is not necessarily determinative. Additionalfactors, such as the binding of other proteins, pH, ion concentration,interaction with other cellular constituents, and the like, can alsoaffect the state of the cellular constituent.

In some embodiments, the activation levels of a plurality ofintracellular activatable elements in single cells are determined. Insome embodiments, at least about 2, 3, 4, 5, 6, 7, 8, 9, 10 or more than10 intracellular activatable elements are determined.

Activation states of activatable elements may result from chemicaladditions or modifications of biomolecules and include biochemicalprocesses such as glycosylation, phosphorylation, acetylation,methylation, biotinylation, glutamylation, glycylation, hydroxylation,isomerization, prenylation, myristoylation, lipoylation,phosphopantetheinylation, sulfation, ISGylation, nitrosylation,palmitoylation, SUMOylation, ubiquitination, neddylation,citrullination, amidation, and disulfide bond formation, disulfide bondreduction. Other possible chemical additions or modifications ofbiomolecules include the formation of protein carbonyls, directmodifications of protein side chains, such as o-tyrosine, chloro-,nitrotyrosine, and dityrosine, and protein adducts derived fromreactions with carbohydrate and lipid derivatives. Other modificationsmay be non-covalent, such as binding of a ligand or binding of anallosteric modulator.

Examples of proteins that may include activatable elements include, butare not limited to kinases, phosphatases, lipid signaling molecules,adaptor/scaffold proteins, cytokines, cytokine regulators,ubiquitination enzymes, adhesion molecules, cytoskeletal/contractileproteins, heterotrimeric G proteins, small molecular weight GTPases,guanine nucleotide exchange factors, GTPase activating proteins,caspases, proteins involved in apoptosis (e.g. PARP), cell cycleregulators, molecular chaperones, metabolic enzymes, vesicular transportproteins, hydroxylases, isomerases, deacetylases, methylases,demethylases, tumor suppressor genes, proteases, ion channels, moleculartransporters, transcription factors/DNA binding factors, regulators oftranscription, and regulators of translation. Examples of activatableelements, activation states and methods of determining the activationlevel of activatable elements are described in US Publication Number20060073474 entitled “Methods and compositions for detecting theactivation state of multiple proteins in single cells” and U.S. Pat. No.7,393,656 entitled “Methods and compositions for risk stratification”the content of which are incorporate here by reference.

In some embodiments, the protein is selected from the group consistingof HER receptors, PDGF receptors, Kit receptor, FGF receptors, Ephreceptors, Trk receptors, IGF receptors, Insulin receptor, Met receptor,Ret, VEGF receptors, TIE1, TIE2, FAK, Jak1, Jak2, Jak3, Tyk2, Src, Lyn,Fyn, Lck, Fgr, Yes, Csk, Abl, Btk, ZAP70, Syk, IRAKs, cRaf, ARaf, BRAF,Mos, Lim kinase, ILK, Tpl, ALK, TGFβ receptors, BMP receptors, MEKKs,ASK, MLKs, DLK, PAKs, Mek 1, Mek 2, MKK3/6, MKK4/7, ASK1,Cot, NIK, Bub,Myt 1, Weel, Casein kinases, PDK1, SGK1, SGK2, SGK3, Akt1, Akt2, Akt3,p90Rsks, p70S6Kinase, Prks, PKCs, PKAs, ROCK 1, ROCK 2, Auroras, CaMKs,MNKs, AMPKs, MELK, MARKs, Chk1, Chk2, LKB-1, MAPKAPKs, Pim1, Pim2, Pim3,IKKs, Cdks, Jnks, Erks, IKKs, GSK3α, GSK3β, Cdks, CLKs, PKR, PI3-Kinaseclass 1, class 2, class 3, mTor, SAPK/JNK1,2,3, p38s, PKR, DNA-PK, ATM,ATR, Receptor protein tyrosine phosphatases (RPTPs), LAR phosphatase,CD45, Non receptor tyrosine phosphatases (NPRTPs), SHPs, MAP kinasephosphatases (MKPs), Dual Specificity phosphatases (DUSPs), CDC25phosphatases, Low molecular weight tyrosine phosphatase, Eyes absent(EYA) tyrosine phosphatases, Slingshot phosphatases (SSH), serinephosphatases, PP2A, PP2B, PP2C, PP1, PP5, inositol phosphatases, PTEN,SHIPs, myotubularins, phosphoinositide kinases, phospholipases,prostaglandin synthases, 5-lipoxygenase, sphingosine kinases,sphingomyelinases, adaptor/scaffold proteins, Shc, Grb2, BLNK, LAT, Bcell adaptor for PI3-kinase (BCAP), SLAP, Dok, KSR, MyD88, Crk, CrkL,GAD, Nck, Grb2 associated binder (GAB), Fas associated death domain(FADD), TRADD, TRAF2, RIP, T-Cell leukemia family, IL-2, IL-4, IL-8,IL-6, interferon γ, interferon α, suppressors of cytokine signaling(SOCs), Cbl, SCF ubiquitination ligase complex, APC/C, adhesionmolecules, integrins, Immunoglobulin-like adhesion molecules, selectins,cadherins, catenins, focal adhesion kinase, p130CAS, fodrin, actin,paxillin, myosin, myosin binding proteins, tubulin, eg5/KSP, CENPs,β-adrenergic receptors, muscarinic receptors, adenylyl cyclasereceptors, small molecular weight GTPases, H-Ras, K-Ras, N-Ras, Ran,Rac, Rho, Cdc42, Arfs, RABs, RHEB, Vav, Tiam, Sos, Dbl, PRK, TSC1,2,Ras-GAP, Arf-GAPs, Rho-GAPs, caspases, Caspase 2, Caspase 3, Caspase 6,Caspase 7, Caspase 8, Caspase 9, PARP, Bcl-2, Mcl-1, Bcl-XL, Bcl-w,Bcl-B, A1, Bax, Bak, Bok, Bik, Bad, Bid, Bim, Bmf, Hrk, Noxa, Puma,IAPB, XIAP, Smac, Cdk4, Cdk 6, Cdk 2, Cdk1, Cdk 7, Cyclin D, Cyclin E,Cyclin A, Cyclin B, Rb, p16, p14Arf, p27KIP, p21CIP, molecularchaperones, Hsp90s, Hsp70, Hsp27, metabolic enzymes, Acetyl-CoAaCarboxylase, ATP citrate lyase, nitric oxide synthase, caveolins,endosomal sorting complex required for transport (ESCRT) proteins,vesicular protein sorting (Vsps), hydroxylases, prolyl-hydroxylasesPHD-1, 2 and 3, asparagine hydroxylase FIH transferases, Pin1 prolylisomerase, topoisomerases, deacetylases, Histone deacetylases, sirtuins,histone acetylases, CBP/P300 family, MYST family, ATF2, DNA methyltransferases, Histone H3K4 demethylases, H3K27, JHDM2A, UTX, VHL, WT-1,p53, Hdm, PTEN, ubiquitin proteases, urokinase-type plasminogenactivator (uPA) and uPA receptor (uPAR) system, cathepsins,metalloproteinases, esterases, hydrolases, separase, potassium channels,sodium channels, multi-drug resistance proteins, P-Gycoprotein,nucleoside transporters, Ets, Elk, SMADs, Rel-A (p65-NFB), CREB, NFAT,ATF-2, AFT, Myc, Fos, Sp1, Egr-1, T-bet, β-catenin, HIFs, FOXOs, E2Fs,SRFs, TCFs, Egr-1, FOXO STAT1, STAT 3, STAT 4, STAT 5, STAT 6, p53,WT-1, HMGA, pS6, 4EPB-1, eIF4E-binding protein, RNA polymerase,initiation factors, elongation factors.

In some embodiments, the classification of a cell according toactivation level of an activatable element, e.g., in a cellular pathwaycomprises classifying the cell as a cell that is correlated with aclinical outcome. In some embodiments, the clinical outcome is theprognosis and/or diagnosis of a condition. In some embodiments, theclinical outcome is the presence or absence of a neoplastic or ahematopoietic condition. In some embodiments, the clinical outcome isthe staging or grading of a neoplastic or hematopoietic condition.Examples of staging include, but are not limited to, aggressive,indolent, benign, refractory, Roman Numeral staging, TNM Staging, Raistaging, Binet staging, WHO classification, FAB classification, IPSSscore, WPSS score, limited stage, extensive stage, staging according tocellular markers such as ZAP70, IgV_(H) mutational status and CD38,occult, including information that may inform on time to progression,progression free survival, overall survival, or event-free survival.

In some embodiments, methods and compositions are provided for theclassification of a cell according to the activation level of anactivatable element, e.g., in a cellular pathway wherein theclassification comprises classifying a cell as a cell that is correlatedto a patient response to a treatment. In some embodiments, the patientresponse is selected from the group consisting of complete response,partial response, nodular partial response, no response, progressivedisease, stable disease and adverse reaction.

In some embodiments, methods and compositions are provided for theclassification of a cell according to the activation level of anactivatable element, e.g., in a cellular pathway wherein theclassification comprises classifying the cell as a cell that iscorrelated with minimal residual disease or emerging resistance.

In some embodiments, methods and compositions are provided for theclassification of a cell according to the activation level of anactivatable element, e.g., in a cellular pathway wherein theclassification comprises selecting a method of treatment. Example ofmethods of treatments include, but are not limited to, chemotherapy,biological therapy, radiation therapy, bone marrow transplantation,Peripheral stem cell transplantation, umbilical cord bloodtransplantation, autologous stem cell transplantation, allogeneic stemcell transplantation, syngeneic stem cell transplantation, surgery,induction therapy, maintenance therapy, and watchful waiting.

Generally, the methods of the invention involve determining theactivation levels of an activatable element in a plurality of singlecells in a sample.

A. Signaling Pathways

In some embodiments, the methods of the invention are employed todetermine the status of an activatable element in a signaling pathway.In some embodiments, a cell is classified, as described herein,according to the activation level of one or more activatable elements inone or more signaling pathways. Signaling pathways and their membershave been extensively described. See (Hunter T. Cell (2000) 100(1):13-27). Exemplary signaling pathways include the following pathways andtheir members: The MAP kinase pathway including Ras, Raf, MEK, ERK andelk; the PI3K/Akt pathway including PI-3-kinase, PDK1, Akt and Bad; theNF-κB pathway including IKKs, IkB and NF-κB and the Wnt pathwayincluding frizzled receptors, beta-catenin, APC and other co-factors andTCF (see Cell Signaling Technology, Inc. 2002 Catolog pages 231-279 andHunter T., supra.). In some embodiments of the invention, the correlatedactivatable elements being assayed (or the signaling proteins beingexamined) are members of the MAP kinase, Akt, NFkB, WNT, STAT and/or PKCsignaling pathways. The methods of the invention also comprise themethods, signaling pathways and signaling molecules disclosed in U.S.61/085,789 which is hereby incorporated by reference in its entirety.

In some embodiments, the methods of the invention are employed todetermine the status of a signaling protein in a signaling pathway knownin the art including those described herein. Exemplary types ofsignaling proteins within the scope of the present invention include,but are not limited to, kinases, kinase substrates (i.e. phosphorylatedsubstrates), phosphatases, phosphatase substrates, binding proteins(such as 14-3-3), receptor ligands and receptors (cell surface receptortyrosine kinases and nuclear receptors)). Kinases and protein bindingdomains, for example, have been well described (see, e.g., CellSignaling Technology, Inc., 2002 Catalogue “The Human Protein Kinases”and “Protein Interaction Domains” pgs. 254-279).

Exemplary signaling proteins include, but are not limited to, kinases,HER receptors, PDGF receptors, Kit receptor, FGF receptors, Ephreceptors, Trk receptors, IGF receptors, Insulin receptor, Met receptor,Ret, VEGF receptors, TIE1, TIE2, FAK, Jak1, Jak2, Jak3, Tyk2, Src, Lyn,Fyn, Lck, Fgr, Yes, Csk, Abl, Btk, ZAP70, Syk, IRAKs, cRaf, ARaf, BRAF,Mos, Lim kinase, ILK, Tpl, ALK, TGFIβ receptors, BMP receptors, MEKKs,ASK, MLKs, DLK, PAKs, Mek 1, Mek 2, MKK3/6, MKK4/7, ASK1, Cot, NIK, Bub,Myt 1, Weel, Casein kinases, PDK1, SGK1, SGK2, SGK3, Akt1, Akt2, Akt3,p90Rsks, p70S6Kinase, Prks, PKCs, PKAs, ROCK 1, ROCK 2, Auroras, CaMKs,MNKs, AMPKs, MELK, MARKs, Chk1, Chk2, LKB-1, MAPKAPKs, Pim1, Pim2, Pim3,IKKs, Cdks, Jnks, Erks, IKKs, GSK3α, GSK3β, Cdks, CLKs, PKR, PI3-Kinaseclass 1, class 2, class 3, mTor, SAPK/JNK1,2,3, p38s, PKR, DNA-PK, ATM,ATR, phosphatases, Receptor protein tyrosine phosphatases (RPTPs), LARphosphatase, CD45, Non receptor tyrosine phosphatases (NPRTP5), SHPs,MAP kinase phosphatases (MKPs), Dual Specificity phosphatases (DUSPs),CDC25 phosphatases, low molecular weight tyrosine phosphatase, Eyesabsent (EYA) tyrosine phosphatases, Slingshot phosphatases (SSH), serinephosphatases, PP2A, PP2B, PP2C, PP1, PP5, inositol phosphatases, PTEN,SHIPs, myotubularins, lipid signaling, phosphoinositide kinases,phospholipases, prostaglandin synthases, 5-lipoxygenase, sphingosinekinases, sphingomyelinases, adaptor/scaffold proteins, Shc, Grb2, BLNK,LAT, B cell adaptor for PI3-kinase (BCAP), SLAP, Dok, KSR, MyD88, Crk,CrkL, GAD, Nck, Grb2 associated binder (GAB), Fas associated deathdomain (FADD), TRADD, TRAF2, RIP, T-Cell leukemia family, cytokines,IL-2, IL-4, IL-8, IL-6, interferon γ, interferon α, cytokine regulators,suppressors of cytokine signaling (SOCs), ubiquitination enzymes, Cbl,SCF ubiquitination ligase complex, APC/C, adhesion molecules, integrins,Immunoglobulin-like adhesion molecules, selectins, cadherins, catenins,focal adhesion kinase, p130CAS, cytoskeletal/contractile proteins,fodrin, actin, paxillin, myosin, myosin binding proteins, tubulin,eg5/KSP, CENPs, heterotrimeric G proteins, β-adrenergic receptors,muscarinic receptors, adenylyl cyclase receptors, small molecular weightGTPases, H-Ras, K-Ras, N-Ras, Ran, Rac, Rho, Cdc42, Arfs, RABs, RHEB,guanine nucleotide exchange factors, Vav, Tiam, Sos, Dbl, PRK, TSC1,2,GTPase activating proteins, Ras-GAP, Arf-GAPs, Rho-GAPs, caspases,Caspase 2, Caspase 3, Caspase 6, Caspase 7, Caspase 8, Caspase 9, PARP,proteins involved in apoptosis, Bcl-2, Mcl-1, Bcl-XL, Bcl-w, Bcl-B, A1,Bax, Bak, Bok, Bik, Bad, Bid, Bim, Bmf, Hrk, Noxa, Puma, IAPB, XIAP,Smac, cell cycle regulators, Cdk4, Cdk 6, Cdk 2, Cdk1, Cdk 7, Cyclin D,Cyclin E, Cyclin A, Cyclin B, Rb, p16, p14Arf, p27KIP, p21CIP, molecularchaperones, Hsp90s, Hsp70, Hsp27, metabolic enzymes, Acetyl-CoAaCarboxylase, ATP citrate lyase, nitric oxide synthase, vesiculartransport proteins, caveolins, endosomal sorting complex required fortransport (ESCRT) proteins, vesicular protein sorting (Vsps),hydroxylases, prolyl-hydroxylases PHD-1, 2 and 3, asparagine hydroxylaseFIH transferases, isomerases, Pin1 prolyl isomerase, topoisomerases,deacetylases, Histone deacetylases, sirtuins, acetylases, histoneacetylases, CBP/P300 family, MYST family, ATF2, methylases, DNA methyltransferases, demethylases, Histone H3K4 demethylases, H3K27, JHDM2A,UTX, tumor suppressor genes, VHL, WT-1, p53, Hdm, PTEN, proteases,ubiquitin proteases, urokinase-type plasminogen activator (uPA) and uPAreceptor (uPAR) system, cathepsins, metalloproteinases, esterases,hydrolases, separase, ion channels, potassium channels, sodium channels,molecular transporters, multi-drug resistance proteins, P-Gycoprotein,nucleoside transporters, transcription factors/DNA binding proteins,Ets, Elk, SMADs, Rel-A (p65-NFKB), CREB, NFAT, ATF-2, AFT, Myc, Fos,Sp1, Egr-1, T-bet, HIFs, FOXOs, E2Fs, SRFs, TCFs, Egr-1, β-catenin, FOXOSTAT1, STAT 3, STAT 4, STAT 5, STAT 6, p53, WT-1, HMGA, regulators oftranslation, pS6, 4EPB-1, eIF4E-binding protein, regulators oftranscription, RNA polymerase, initiation factors, and elongationfactors.

In some embodiments the protein is selected from the group consisting ofPI3-Kinase (p85, p110a, p110b, p110d), Jak1, Jak2, SOCs, Rac, Rho,Cdc42, Ras-GAP, Vav, Tiam, Sos, Dbl, Nck, Gab, PRK, SHPT, and SHP2,SHIP1, SHIP2, sSHIP, PTEN, Shc, Grb2, PDK1, SGK, Akt1, Akt2, Akt3,TSC1,2, Rheb, mTor, 4EBP-1, p70S6Kinase, S6, LKB-1, AMPK, PFK,Acetyl-CoAa Carboxylase, DokS, Rafs, Mos, Tpl2, MEK1/2, MLK3, TAK, DLK,MKK3/6, MEKK1,4, MLK3, ASK1, MKK4/7, SAPK/JNK1,2,3, p38s, Erk1/2, Syk,Btk, BLNK, LAT, ZAP70, Lck, Cbl, SLP-76, PLCγ₁, PLCγ₂, STAT1, STAT 3,STAT 4, STAT 5, STAT 6, FAK, p130CAS, PAKs, LIMK1/2, Hsp90, Hsp70,Hsp27, SMADs, Rel-A (p65-NFKB), CREB, Histone H₂B, HATs, HDACs, PKR, Rb,Cyclin D, Cyclin E, Cyclin A, Cyclin B, P16, p14Arf, p27KIP, p21CIP,Cdk4, Cdk6, Cdk7, Cdk1, Cdk2, Cdk9, Cdc25,A/B/C, Abl, E2F, FADD, TRADD,TRAF2, RIP, Myd88, BAD, Bcl-2, Mcl-1, Bcl-XL, Caspase 2, Caspase 3,Caspase 6, Caspase 7, Caspase 8, Caspase 9, PARP, IAPB, Smac, Fodrin,Actin, Src, Lyn, Fyn, Lck, NIK, IKB, p65(ReiA), IKKα, PKA, PKCα, PKCβ,PKCθ, PKCδ, CAMK, Elk, AFT, Myc, Egr-1, NFAT, ATF-2, Mdm2, p53, DNA-PK,Chk1, Chk2, ATM, ATR, β-catenin, CrkL, GSK3α, GSK3β, and FOXO.

MAP kinase pathway: In some embodiments, the methods of the inventionare employed to determine the status of an activatable element in theMAP kinase pathway. Without intending to be limited to any theory, theMAP Kinase pathway is a signal transduction pathway that couplesintracellular responses to the binding of growth factors to cell surfacereceptors. This pathway is very complex and includes many proteincomponents. In many cell types, activation of this pathway promotes celldivision.

Receptor-linked tyrosine kinases such as the epidermal growth factorreceptor (EGFR) are activated by extracellular ligands. Binding ofepidermal growth factor (EGF) to the EGFR activates the tyrosine kinaseactivity of the cytoplasmic domain of the receptor. The EGFR becomesphosphorylated on tyrosines. Docking proteins such as GRB2 contain SH2domains that bind to the phosphotyrosines of the activated receptor.GRB2 binds to the guanine nucleotide exchange factor SOS by way of anSH3 domain of GRB2. When the GRB2-SOS complex docks to phosphorylatedEGFR, SOS becomes activated. Activated SOS promotes the removal of GDPfrom Ras. Ras can then bind GTP and become active. Other small Gproteins can be activated in a similar way, but are not discussedfurther here. Activated Ras activates the protein kinase activity of RAFkinase, a serine/threonine-selective protein kinase. RAF kinasephosphorylates and activates MEK, another serine/threonine kinase. MEKphosphorylates and activates mitogen-activated protein kinase (MAPK).

Technically, RAF, MEK and MAPK are all mitogen-activated kinases, as isMNK. MAPK was originally called “extracellular signal-regulated kinases”(ERKs) and microtubule-associated protein kinase (MAPK). One of thefirst proteins known to be phosphorylated by ERK was amicrotubule-associated protein. Many additional targets forphosphorylation by MAPK have been found and the protein was re-named“mitogen-activated protein kinase” (MAPK). The series of kinases fromRAF to MEK to MAPK is an example of a protein kinase cascade. Suchseries of kinases provide opportunities for feedback regulation andsignal amplification. RAS is activated in a wide range of cancers (seeCell Signaling Technology, Inc. Catolog, supra. at pages 231-279 andHunter T, supra. and references therein).

PI3K/Akt pathway: In some embodiments, the methods of the invention areemployed to determine the status of an activatable element in a PI3K/Aktpathway. Without intending to be limited to any theory, the PI3K/Aktpathway plays a role in effecting alterations in abroad range ofcellular functions in response to extracellular signals. A downstreameffector of PI3K is the serine-threonine kinase Akt which in response toPI3K activation, phosphorylates and regulates the activity of a numberof targets including kinases, transcription factors and other regulatorymolecules. The serine/threonine kinase Akt functions intracellularly asa nodal point for a constellation of converging upstream signalingpathways, which involve stimulation of receptor tyrosine kinases such asIGF-1R, HER2/Neu, VEGF-R, PDGF-R), and an assembly of membrane-localizedcomplexes of receptor-PI3K and activation of Akt through the secondmessenger PIP3. The integration of these intracellular signals at thelevel of Akt and its kinase activity, regulates the phosphorylation ofits several downstream effectors, such as NF-B, mTOR, Forkhead, Bad,GSK-3 and MDM-2. These phosphorylation events, in turn, mediate theeffects of Akt on cell growth, proliferation, protection frompro-apoptotic stimuli, and stimulation of neoangiogenesis. Akt and itsupstream regulators are deregulated in a wide range of solid tumors andhematologic malignancies. The Akt pathway is the central cell survivalpathway that is activated by such oncogenic events as over expression ofan upstream receptor tyrosine kinase such as EGFR (ibid) or loss of anupstream regulatory protein such as PTEN (ibid).

NE-κB pathway: In some embodiments, the methods of the invention areemployed to determine the status of an activatable element in a NF-κBpathway. Without intending to be limited to any theory, the NF-κBpathway is involved in regulating many aspects of cellular activity, instress, injury and especially in pathways of the immune response. Someexamples are the response to and induction of IL-2, the induction ofTAP1 and MHC molecules by NF-κB, and many aspects of the inflammatoryresponse, e.g. induction of IL-1 (alpha and beta), TNF-alpha andleukocyte adhesion molecules (E-selectin, VCAM-1 and ICAM-1). Moreover,NF-κB is involved in many aspects of cell growth, differentiation andproliferation via the induction of certain growth and transcriptionfactors (e.g. c-myc, ras and p53). The NF-κB signal transduction pathwayis misregulated in a variety of human cancers, especially those oflymphoid cell origin. Several human lymphoid cancer cells are reportedto have mutations or amplifications of genes encoding NF-κBtranscription factors. In most cancer cells NF-κB is constitutivelyactive and resides in the nucleus. In some cases, this may be due tochronic stimulation of the IKK pathway, while in others the geneencoding IkBa may be defective. Such continuous nuclear NF-κB activitynot only protects cancer cells from apoptotic cell death, but may evenenhance their growth activity. Designing anti-tumor agents to blockNF-κB activity or to increase their sensitivity to conventionalchemotherapy may have great therapeutic value.

WNT pathway: In some embodiments, the methods of the invention areemployed to determine the status of an activatable element in a WNTpathway. Without intending to be limited to any theory, the Wntsignaling pathway describes a complex network of proteins most wellknown for their roles in embryogenesis and cancer, but also involved innormal physiological processes in adult animals. The canonical Wntpathway describes a series of events that occur when Wnt proteins bindto cell-surface receptors of the Frizzled family, causing the receptorsto activate Dishevelled family proteins and ultimately resulting in achange in the amount of β-catenin that reaches the nucleus. Dishevelled(DSH) is a key component of a membrane-associated Wnt receptor complexwhich, when activated by Wnt binding, inhibits a second complex ofproteins that includes axin, GSK-3, and the protein APC. Theaxin/GSK-3/APC complex normally promotes the proteolytic degradation ofthe β-catenin intracellular signaling molecule. After this “β-catenindestruction complex” is inhibited, a pool of cytoplasmic β-cateninstabilizes, and some β-catenin is able to enter the nucleus and interactwith TCF/LEF family transcription factors to promote specific geneexpression.

PKC pathway: In some embodiments, the methods of the invention areemployed to determine the status of an activatable element in a PKCpathway. Without intending to be limited to any theory, PKC pathway isassociated with cell proliferation, differentiation, and apoptosis. Atleast eleven closely related PKC isozymes have been reported that differin their structure, biochemical properties, tissue distribution,subcellular localization, and substrate specificity. They are classifiedas conventional (α, β1, β2, γ), novel (δ, ε, η, θ, μ), and atypical (ζ,λ) isozymes. Conventional PKC isozymes are Ca2+-dependent, while noveland atypical isozymes do not require Ca2+ for their activation. All PKCisozymes, with the exception of ζ and λ, are activated by diacylglycerol(DAG). PKC isozymes negatively or positively regulate critical cellcycle transitions, including cell cycle entry and exit and the G1 and G2checkpoints. Altered PKC activity has been linked with various types ofmalignancies. Higher levels of PKC and differential activation ofvarious PKC isozymes have been reported in breast tumors, adenomatouspituitaries, thyroid cancer tissue, leukemic cells, and lung cancercells. Down regulation of PKCα is reported in the majority of colonadenocarcinomas and in the early stages of intestinal carcinogenesis.Thus, PKC inhibitors have become important tools in the treatment ofcancers. The involvement of PKC in the regulation of apoptosis addsanother dimension to the effort to develop drugs that will specificallytarget PKC. PKC pathway activation is thought to also play a role indiseases such as cardiovascular disease and diabetes.

In some embodiments of the invention, the methods described herein areemployed to determine the status of an activatable element in asignaling pathway. Methods and compositions are provided for theclassification of a cell according to the status of an activatableelement in a signaling pathway. The cell can be a hematopoietic cell.Examples of hematopoietic cells include but are not limited topluripotent hematopoietic stem cells, B-lymphocyte lineage progenitor orderived cells, T-lymphocyte lineage progenitor or derived cells, NK celllineage progenitor or derived cells, granulocyte lineage progenitor orderived cells, monocyte lineage progenitor or derived cells,megakaryocyte lineage progenitor or derived cells and erythroid lineageprogenitor or derived cells.

In some embodiments, the classification of a cell according to thestatus of an activatable element in a signaling pathway comprisesclassifying the cell as a cell that is correlated with a clinicaloutcome. In some embodiments, the clinical outcome is the prognosisand/or diagnosis of a condition. In some embodiments, the clinicaloutcome is the presence or absence of a neoplastic or a hematopoieticcondition. In some embodiments, the clinical outcome is the staging orgrading of a neoplastic or hematopoietic condition. Examples of staginginclude, but are not limited to, aggressive, indolent, benign,refractory, Roman Numeral staging, TNM Staging, Rai staging, Binetstaging, WHO classification, FAB classification, IPSS score, WPSS score,limited stage, extensive stage, staging according to cellular markerssuch as ZAP70, IgV_(H) mutational status and CD38, occult, includinginformation that may inform on time to progression, progression freesurvival, overall survival, or event-free survival.

In some embodiments, methods and compositions are provided for theclassification of a cell according to the status of an activatableelement in a signaling pathway wherein the classification comprisesclassifying a cell as a cell that is correlated to a patient response toa treatment. In some embodiments, the patient response is selected fromthe group consisting of complete response, partial response, nodularpartial response, no response, progressive disease, stable disease andadverse reaction.

In some embodiments, methods and compositions are provided for theclassification of a cell according to the status of an activatableelement in a signaling pathway wherein the classification comprisesclassifying the cell as a cell that is correlated with minimal residualdisease or emerging resistance.

In some embodiments, methods and compositions are provided for theclassification of a cell according to the status of an activatableelement in a signaling pathway wherein the classification comprisesselecting a method of treatment. Example of methods of treatmentsinclude, but are not limited to, chemotherapy, biological therapy,radiation therapy, bone marrow transplantation, Peripheral stem celltransplantation, umbilical cord blood transplantation, autologous stemcell transplantation, allogeneic stem cell transplantation, syngeneicstem cell transplantation, surgery, induction therapy, maintenancetherapy, watchful waiting, and holistic/alternative therapy.

The invention is not limited to presently elucidated signaling pathwaysand signal transduction proteins, and encompasses signaling pathways andproteins subsequently identified.

B. B-Cell Receptor Pathway

In some embodiments, the methods and compositions of the invention maybe employed to examine and profile the status of any activatable elementin B-Cell Receptor (BCR) signaling, or collections of such activatableelements in a B-lymphocyte lineage progenitor or derived cell. In someembodiments, the physiological status of one or more B-lymphocytelineage progenitor or derived cell is determined by examining andprofiling the status of one or more activatable element in BCRsignaling. In some embodiments, a B-lymphocyte lineage progenitor orderived cell is classified, as described herein, according to theactivation level of one or more activatable elements in BCR signaling.Examples of B-lymphocyte lineage derived cell include, but are notlimited to, B-lymphocyte lineage early pro-B cell, late pro-B cell,large pre-B cell, small pre-B cell, immature B cell, mature B cell,plasma cell, memory B cell, a CD5+ B cell, a CD38+ B cell, a B cellbearing a mutatated or non mutated heavy chain of the B cell receptorand a B cell expressing Zap70. In some embodiments, the B-lymphocytelineage progenitor or derived cell is a cell associated with a conditionas described herein.

Without intending to be limited to any theory, BCR cross-linkingtriggers phosphorylation of tyrosines within the ITAM motif domains ofIgα and Igβ by Src family member tyrosine kinases (e.g., Lyn, Lck, Blk,Fyn). The phosphorylated ITAMs of Igαβ recruit and enhancephosphorylation of Syk (directly) and Btk (via Syk). BCR cross-linkingalso brings together numerous regulator and adapter molecules (e.g.,SLP-65/BLNK, Grb2, CD22, SHP-1) and compartmentalizes the BCR in lipidrafts with coreceptors CD19 and CD21. Following Syk and Btk activation,the enzymes phospholipase-Cγ2 (PLC γ₂) and PI3K propagate BCR signaling.PLC γ₂ activation generates calcium flux, inositol-1,4,5-triphosphate,and diacylglycerol, and results in activation of protein kinase C andNF-κB. Syk interacts with PLC γ₂ via adapters, whereas Btk can interactdirectly, and each is required for PLC γ₂ activity following BCRcross-linking Both Syk and Btk can activate PI3K following BCRcross-linking Activation of PI3K enables Akt-mediated survivalsignaling, and PI3K is required for BCR-mediated survival during B celldevelopment. PLC γ₂ and PI3K also initiate kinase cascades that resultin phosphorylation of the MAPK family proteins ERK1/2 and p38.Activation of the Ras-Raf-ERK1/2 signaling cascade is considered acentral event in BCR signaling, and decreased Ras activation due toRasGRP1 and RasGRP3 loss in mouse impairs B cell proliferation. Incontrast, p38 is a stress response protein that interacts with p53 andregulates cell cycle checkpoints. Differential activation of ERK1/2 andp38 might enable the BCR to drive diverse cellular outcomes, but thequestion arises whether a given B cell activates these two pathwayssimultaneously or favors one pathway depending on additional signalingcontext.

Efficient activation of BCR signaling depends on generation of H₂O₂ andinactivation of negative regulatory protein tyrosine phosphatases(PTPs). Following BCR cross-linking, recruitment and activation ofcalcium-dependent NADPH oxidases (NOX) proteins, such as NOX5, enablesproduction of H₂O₂ and lowers the signaling threshold for the BCR.BCR-induced H₂O₂ transiently inactivates membrane proximal PTPs,including SHP-1, via reversible oxidation of the catalytic cysteine tosulfenic acid. Elegant work reconstituting the BCR signaling pathway ininsect cells has suggested a model of redox feedback loops where H₂O₂inactivates PTPs and enables amplification of early signaling events,such as Syk phosphorylation and ITAM binding. Recent work characterizedendogenously generated H₂O₂ as the primary redox species generated byBCR signaling and indicated that NOX-dependent production of H₂O₂ wascritical to initiate a wave of BCR signaling in mouse A20 B cells.

In some embodiments, the invention provides a method for classifying aB-lymphocyte lineage progenitor or derived cell upon treatment with amodulator and/or inhibitor. Examples of B-lymphocyte lineage progenitoror derived cells include, but are not limited to an early pro-B cell,late pro-B cell, large pre-B cell, small pre-B cell, immature B cell,mature B cell, plasma cell and memory B cell, a CD5+ B cell, a CD38+ Bcell, a B cell bearing a mutatated or non mutated heavy chain of the Bcell receptor, or a B cell expressing Zap70.

In some embodiments, the classification includes classifying the cellaccording to the status of an activatable element in a BCR pathway as acell that is correlated with a clinical outcome. In some embodiments,the invention provides methods for classifying a B-lymphocyte lineageprogenitor or derived cell based on an alteration in signaling proximalto the BCR. In some embodiments, the clinical outcome is the prognosisand/or diagnosis of a condition. In some embodiments, the clinicaloutcome is the presence or absence of a neoplastic or a hematopoieticcondition, such as Chronic Lymphocytic Leukemia (CLL), B lymphocytelineage leukemia, B lymphocyte lineage lymphoma, Multiple Myeloma, orplasma cell disorders, e.g., amyloidosis or Waldenstrom'smacroglobulinemia. In some embodiments, the condition is CLL. In someembodiments, the invention provides methods for classifying a CLL cellbased on an alteration in signaling proximal to the BCR. The presence ofthe alteration is indicative of a clinical outcome. In some embodimentsCLL is defined by a monoclonal B cell population that co-expresses CD5with CD19 and CD23 or CD5 with CD20 and CD23 and by surfaceimmunoglobulin expression. In some embodiments, CLL is defined by amonoclonal B cell population that co-expresses CD5 with CD19 and CD23 orCD5 with CD20 and CD23 and dim surface immunoglobulin expression.Additional B-cell markers can be used to identify or classify aB-lymphocyte lineage progenitor or derived cell. Non-limiting examplesof such markers include CD45, CD5, CD14, CD19, CD20, CD22, CD23, CD27,CD37, CD40, CD52, CD79, CD38, CD96, major histocompatability antigen(MHC) Class1 and MHC Class 2.

In some embodiments, the clinical outcome is the staging or grading of aneoplastic or hematopoietic condition. Examples of staging in methodsprovided by the invention include aggressive, indolent, benign,refractory, Roman Numeral staging, TNM Staging, Rai staging, Binetstaging, WHO classification, FAB classification, IPSS score, WPSS score,limited stage, extensive stage, staging according to cellular markerssuch as ZAP70, IgV_(H) mutational status and CD38, occult, includinginformation that may inform on time to progression, progression freesurvival, overall survival, or event-free survival.

In some embodiments of the methods of the invention, the classifying ofthe B-lymphocyte lineage progenitor or derived cell based on activationlevel of an activatable element in BCR pathway includes classifying thecell as a cell that is correlated to a patient response to a treatment,such as complete response, partial response, nodular partial response,no response, progressive disease, stable disease, relapse or adversereaction. The method may further comprise determining a method oftreatment, e.g., chemotherapy, biological therapy, radiation therapy,bone marrow transplantation, Peripheral stem cell transplantation,umbilical cord blood transplantation, autologous stem celltransplantation, allogeneic stem cell transplantation, syngeneic stemcell transplantation, surgery, induction therapy, maintenance therapy,watchful waiting, or holistic/alternative therapy.

In some embodiments of the methods of the invention, the classifying ofthe B-lymphocyte lineage progenitor or derived cells based on activationof an activatable element in BCR pathway includes classifying the cellas a cell that is correlated with minimal residual disease or emergingresistance.

C. Tonic Signaling

In some embodiments, the methods and compositions of the invention maybe employed to determine the status of a tonic signaling pathway in acell. In some embodiments, the methods and compositions of the inventionmay be employed to examine and profile the status of any activatableelement in a tonic signaling pathway, or collections of such activatableelements in a cell. In some embodiments, the physiological status of acell is determined by examining and profiling the status of one or moreactivatable elements in a tonic signaling pathway. In some embodiments,a cell is classified, as described herein, according to the status ofone or more activatable elements in a tonic signaling pathway. The term“tonic signaling” includes antigen-independent signaling, independentbasal signaling and non-induced or ligand-independent signaling.

Without intending to be limited to any theory, recent evidence supportsthe notion that in most signal transduction systems regulated bycellular receptors some basal level of signaling occurs continuously ina ligand-independent manner, although the flux through such systems mayvary considerably. The basal tone or the steady state level of signalingin unstimulated cells is the result of equilibrium of positive andnegative regulators within a signaling pathway. Thus, the balancedactions of positive and negative regulators of signal transduction setthe steady state equilibrium. Receptor stimulation then perturbs theequilibrium state in various ways to initiate cellular responses. Thesteady state level of signaling in the unstimulated state may itselfhave functional consequences, for instance, to maintain certaindifferentiated cellular properties or functions.

In some embodiments, the invention provides for methods of determiningtonic signaling status of a cell. In some embodiments, the tonicsignaling is a cellular receptor tonic signaling. In some embodiments,the tonic signaling is a BCR tonic signaling. Methods and compositionsare provided for the classification of a cell according to the status ofan activatable element in a tonic signaling pathway. The cell can be ahematopoietic cell. Examples of hematopoietic cells include but are notlimited to pluripotent hematopoietic stem cells, B-lymphocyte lineageprogenitor or derived cells, T-lymphocyte lineage progenitor or derivedcells, NK cell lineage progenitor or derived cells, granulocyte lineageprogenitor or derived cells, monocyte lineage progenitor or derivedcells, megakaryocyte lineage progenitor or derived cells and erythroidlineage progenitor or derived cells.

In some embodiments, the classification of a cell according to thestatus of an activatable element in a tonic signaling pathway comprisesclassifying the cell as a cell that is correlated with a clinicaloutcome. In some embodiments, the clinical outcome is the prognosisand/or diagnosis of a condition. In some embodiments, the clinicaloutcome is the presence or absence of a neoplastic or a hematopoieticcondition. Examples of neoplastic or hematopoietic conditions include,but are not limited to, such as Chronic Lymphocytic Leukemia (CLL), Blymphocyte lineage leukemia, B lymphocyte lineage lymphoma, MultipleMyeloma, or plasma cell disorders, e.g., amyloidosis or Waldenstrom'smacroglobulinemia. In some embodiments, the condition is CLL. In someembodiments, CLL is defined by a monoclonal B cell population thatco-expresses CD5 with CD19 and CD23 or CD5 with CD20 and CD23 and bysurface immunoglobulin expression.

In some embodiments, the clinical outcome is the staging or grading of aneoplastic or hematopoietic condition. Examples of staging include, butare not limited to, aggressive, indolent, benign, refractory, RomanNumeral staging, TNM Staging, Rai staging, Binet staging, WHOclassification, FAB classification, IPSS score, WPSS score, limitedstage, extensive stage, staging according to cellular markers such asZAP70, IgV_(H) mutational status and CD38, including information thatmay inform on time to progression, progression free survival, overallsurvival, or event-free survival.

In some embodiments, the invention provides methods for classifying aCLL cell based on an alteration in signaling proximal to the BCR that isindicative of the presence of tonic signaling. The presence of thealteration is indicative of a clinical outcome, where the clinicaloutcome is as described herein.

In some embodiments, methods and compositions are provided for theclassification of a cell according to the status of an activatableelement in a tonic signaling pathway wherein the classificationcomprises classifying a cell as a cell that is correlated to a patientresponse to a treatment. In some embodiments, the patient response isselected from the group consisting of complete response, partialresponse, nodular partial response, no response, progressive disease,stable disease and adverse reaction.

In some embodiments, methods and compositions are provided for theclassification of a cell according to the status of an activatableelement in a tonic signaling pathway wherein the classificationcomprises classifying the cell as a cell that is correlated with minimalresidual disease or emerging resistance.

In some embodiments, methods and compositions are provided for theclassification of a cell according to the status of an activatableelement in a tonic signaling pathway wherein the classificationcomprises selecting a method of treatment. Example of methods oftreatments include, but are not limited to, chemotherapy, biologicaltherapy, radiation therapy, bone marrow transplantation, Peripheral stemcell transplantation, umbilical cord blood transplantation, autologousstem cell transplantation, allogeneic stem cell transplantation,syngeneic stem cell transplantation, surgery, induction therapy,maintenance therapy, watchful waiting, and holistic/alternative therapy.

Binding Element

In some embodiments of the invention, the activation level of anactivatable element is determined by contacting a cell with a bindingelement that is specific for an activation state of the activatableelement. The term “Binding element” includes any molecule, e.g.,peptide, nucleic acid, small organic molecule which is capable ofdetecting an activation state of an activatable element over anotheractivation state of the activatable element.

In some embodiments, the binding element is a peptide, polypeptide,oligopeptide or a protein. The peptide, polypeptide, oligopeptide orprotein may be made up of naturally occurring amino acids and peptidebonds, or synthetic peptidomimetic structures. Thus “amino acid”, or“peptide residue”, as used herein include both naturally occurring andsynthetic amino acids. For example, homo-phenylalanine, citrulline andnoreleucine are considered amino acids for the purposes of theinvention. The side chains may be in either the (R) or the (S)configuration. In some embodiments, the amino acids are in the (S) orL-configuration. If non-naturally occurring side chains are used,non-amino acid substituents may be used, for example to prevent orretard in vivo degradation. Proteins including non-naturally occurringamino acids may be synthesized or in some cases, made recombinantly; seevan Hest et al., FEBS Lett 428:(1-2) 68-70 May 22, 1998 and Tang et al.,Abstr. Pap Am. Chem. S218: U138 Part 2 Aug. 22, 1999, both of which areexpressly incorporated by reference herein.

Methods of the present invention may be used to detect any particularactivatable element in a sample that is antigenically detectable andantigenically distinguishable from other activatable element which ispresent in the sample. For example, as demonstrated (see, e.g., theExamples) and described herein, the activation state-specific antibodiesof the present invention can be used in the present methods to identifydistinct signaling cascades of a subset or subpopulation of complex cellpopulations; and the ordering of protein activation (e.g., kinaseactivation) in potential signaling hierarchies. Hence, in someembodiments the expression and phosphorylation of one or morepolypeptides are detected and quantified using methods of the presentinvention. In some embodiments, the expression and phosphorylation ofone or more polypeptides that are cellular components of a cellularpathway are detected and quantified using methods of the presentinvention. As used herein, the term “activation state-specific antibody”or “activation state antibody” or grammatical equivalents thereof, referto an antibody that specifically binds to a corresponding and specificantigen. Preferably, the corresponding and specific antigen is aspecific form of an activatable element. Also preferably, the binding ofthe activation state-specific antibody is indicative of a specificactivation state of a specific activatable element.

In some embodiments, the binding element is an antibody. In someembodiment, the binding element is an activation state-specificantibody. In some embodiment, the binding element is an phospho-specificantibody.

The term “antibody” includes full length antibodies and antibodyfragments, and may refer to a natural antibody from any organism, anengineered antibody, or an antibody generated recombinantly forexperimental, therapeutic, or other purposes as further defined below.Examples of antibody fragments, as are known in the art, such as Fab,Fab′, F(ab′)2, Fv, scFv, or other antigen-binding subsequences ofantibodies, either produced by the modification of whole antibodies orthose synthesized de novo using recombinant DNA technologies. The term“antibody” comprises monoclonal and polyclonal antibodies. Antibodiescan be antagonists, agonists, neutralizing, inhibitory, or stimulatory.

The antibodies of the present invention may be nonhuman, chimeric,humanized, or fully human. For a description of the concepts of chimericand humanized antibodies see Clark et al., 2000 and references citedtherein (Clark, (2000) Immunol. Today 21:397-402). Chimeric antibodiescomprise the variable region of a nonhuman antibody, for example VH andVL domains of mouse or rat origin, operably linked to the constantregion of a human antibody (see for example U.S. Pat. No. 4,816,567). Insome embodiments, the antibodies of the present invention are humanized.By “humanized” antibody as used herein is meant an antibody comprising ahuman framework region (FR) and one or more complementarity determiningregions (CDR's) from a non-human (usually mouse or rat) antibody. Thenon-human antibody providing the CDR's is called the “donor” and thehuman immunoglobulin providing the framework is called the “acceptor”.Humanization relies principally on the grafting of donor CDRs ontoacceptor (human) VL and VH frameworks (Winter U.S. Pat. No. 5,225,539).This strategy is referred to as “CDR grafting”. “Backmutation” ofselected acceptor framework residues to the corresponding donor residuesis often required to regain affinity that is lost in the initial graftedconstruct (U.S. Pat. No. 5,530,101; U.S. Pat. No. 5,585,089; U.S. Pat.No. 5,693,761; U.S. Pat. No. 5,693,762; U.S. Pat. No. 6,180,370; U.S.Pat. No. 5,859,205; U.S. Pat. No. 5,821,337; U.S. Pat. No. 6,054,297;U.S. Pat. No. 6,407,213). The humanized antibody optimally also willcomprise at least a portion of an immunoglobulin constant region,typically that of a human immunoglobulin, and thus will typicallycomprise a human Fc region. Methods for humanizing non-human antibodiesare well known in the art, and can be essentially performed followingthe method of Winter and co-workers (Jones et al., 1986, Nature321:522-525; Riechmann et al., 1988, Nature 332:323-329; Verhoeyen etal., 1988, Science, 239:1534-1536). Additional examples of humanizedmurine monoclonal antibodies are also known in the art, for exampleantibodies binding human protein C(O'Connor et al., 1998, Protein Eng11:321-8), interleukin 2 receptor (Queen et al., 1989, Proc Natl AcadSci, USA 86:10029-33), and human epidermal growth factor receptor 2(Carter et al., 1992, Proc Natl. Acad Sci USA 89:4285-9). In analternate embodiment, the antibodies of the present invention may befully human, that is the sequences of the antibodies are completely orsubstantially human. A number of methods are known in the art forgenerating fully human antibodies, including the use of transgenic mice(Bruggemann et al., 1997, Curr Opin Biotechnol 8:455-458) or humanantibody libraries coupled with selection methods (Griffiths et al.,1998, Curr Opin Biotechnol 9:102-108).

Specifically included within the definition of “antibody” areaglycosylated antibodies. By “aglycosylated antibody” as used herein ismeant an antibody that lacks carbohydrate attached at position 297 ofthe Fc region, wherein numbering is according to the EU system as inKabat. The aglycosylated antibody may be a deglycosylated antibody,which is an antibody for which the Fc carbohydrate has been removed, forexample chemically or enzymatically. Alternatively, the aglycosylatedantibody may be a nonglycosylated or unglycosylated antibody, that is anantibody that was expressed without Fc carbohydrate, for example bymutation of one or residues that encode the glycosylation pattern or byexpression in an organism that does not attach carbohydrates toproteins, for example bacteria.

As pointed out above, activation state specific antibodies can be usedto detect kinase activity, however additional means for determiningkinase activation are provided by the present invention. For example,substrates that are specifically recognized by protein kinases andphosphorylated thereby are known. Antibodies that specifically bind tosuch phosphorylated substrates but do not bind to suchnon-phosphorylated substrates (phospho-substrate antibodies) may be usedto determine the presence of activated kinase in a sample.

In a further embodiment, an element activation profile is determinedusing a multiplicity of activation state antibodies that have beenimmobilized. Antibodies may be non-diffusibly bound to an insolublesupport having isolated sample-receiving areas (e.g. a microtiter plate,an array, etc.). The insoluble supports may be made of any compositionto which the compositions can be bound, is readily separated fromsoluble material, and is otherwise compatible with the overall method ofscreening. The surface of such supports may be solid or porous and ofany convenient shape. Examples of suitable insoluble supports includemicrotiter plates, arrays, membranes, and beads. These are typicallymade of glass, plastic (e.g., polystyrene), polysaccharides, nylon ornitrocellulose, Teflon™, etc. Microtiter plates and arrays areespecially convenient because a large number of assays can be carriedout simultaneously, using small amounts of reagents and samples. In somecases magnetic beads and the like are included.

The particular manner of binding of the composition is not crucial solong as it is compatible with the reagents and overall methods of theinvention, maintains the activity of the composition and isnon-diffusible. Methods of binding include the use of antibodies (whichdo not sterically block either the ligand binding site or activationsequence when the protein is bound to the support), direct binding to“sticky” or ionic supports, chemical cross-linking, the synthesis of theantibody on the surface, etc. Following binding of the antibody, excessunbound material is removed by washing. The sample receiving areas maythen be blocked through incubation with bovine serum albumin (BSA),casein or other innocuous protein or other moiety.

The antigenicity of an activated isoform of an activatable element isdistinguishable from the antigenicity of non-activated isoform of anactivatable element or from the antigenicity of an isoform of adifferent activation state. In some embodiments, an activated isoform ofan element possesses an epitope that is absent in a non-activatedisoform of an element, or vice versa. In some embodiments, thisdifference is due to covalent addition of moieties to an element, suchas phosphate moieties, or due to a structural change in an element, asthrough protein cleavage, or due to an otherwise induced conformationalchange in an element which causes the element to present the samesequence in an antigenically distinguishable way. In some embodiments,such a conformational change causes an activated isoform of an elementto present at least one epitope that is not present in a non-activatedisoform, or to not present at least one epitope that is presented by anon-activated isoform of the element. In some embodiments, the epitopesfor the distinguishing antibodies are centered around the active site ofthe element, although as is known in the art, conformational changes inone area of an element may cause alterations in different areas of theelement as well.

Many antibodies, many of which are commercially available (for example,see Cell Signaling Technology, www.cellsignal.com, Millipore,eBioscience, Caltag, Santa Cruz Biotech, Abcam, BD Biosciences, Sigmaand Anaspec) the contents which are incorporated herein by reference)have been produced which specifically bind to the phosphorylated isoformof a protein but do not specifically bind to a non-phosphorylatedisoform of a protein. Many such antibodies have been produced for thestudy of signal transducing proteins which are reversiblyphosphorylated. Particularly, many such antibodies have been producedwhich specifically bind to phosphorylated, activated isoforms ofprotein. Examples of proteins that can be analyzed with the methodsdescribed herein include, but are not limited to, kinases, HERreceptors, PDGF receptors, Kit receptor, FGF receptors, Eph receptors,Trk receptors, IGF receptors, Insulin receptor, Met receptor, Ret, VEGFreceptors, TIE1, TIE2, FAK, Jak1, Jak2, Jak3, Tyk2, Src, Lyn, Fyn, Lck,Fgr, Yes, Csk, Abl, Btk, ZAP70, Syk, IRAKs, cRaf, ARaf, BRAF, Mos, Limkinase, ILK, Tpl, ALK, TGFβ receptors, BMP receptors, MEKKs, ASK, MLKs,DLK, PAKs, Mek 1, Mek 2, MKK3/6, MKK4/7, ASK1, Cot, NIK, Bub, Myt 1,Weel, Casein kinases, PDK1, SGK1, SGK2, SGK3, Akt1, Akt2, Akt3, p90Rsks,p70S6Kinase, Prks, PKCs, PKAs, ROCK 1, ROCK 2, Auroras, CaMKs, MNKs,AMPKs, MELK, MARKs, Chk1, Chk2, LKB-1, MAPKAPKs, Pim1, Pim2, Pim3, IKKs,Cdks, Jnks, Erks, IKKs, GSK3α, GSK3β, Cdks, CLKs, PKR, PI3-Kinase class1, class 2, class 3, mTor, SAPK/JNK1,2,3, p38s, PKR, DNA-PK, ATM, ATR,phosphatases, Receptor protein tyrosine phosphatases (RPTPs), LARphosphatase, CD45, Non receptor tyrosine phosphatases (NPRTPs), SHPs,MAP kinase phosphatases (MKPs), Dual Specificity phosphatases (DUSPs),CDC25 phosphatases, Low molecular weight tyrosine phosphatase, Eyesabsent (EYA) tyrosine phosphatases, Slingshot phosphatases (SSH), serinephosphatases, PP2A, PP2B, PP2C, PP1, PP5, inositol phosphatases, PTEN,SHIPs, myotubularins, lipid signaling, phosphoinositide kinases,phospholipases, prostaglandin synthases, 5-lipoxygenase, sphingosinekinases, sphingomyelinases, adaptor/scaffold proteins, Shc, Grb2, BLNK,LAT, B cell adaptor for PI3-kinase (BCAP), SLAP, Dok, KSR, MyD88, Crk,CrkL, GAD, Nck, Grb2 associated binder (GAB), Fas associated deathdomain (FADD), TRADD, TRAF2, RIP, T-Cell leukemia family, cytokines,IL-2, IL-4, IL-8, IL-6, interferon γ, interferon α, cytokine regulators,suppressors of cytokine signaling (SOCs), ubiquitination enzymes, Cbl,SCF ubiquitination ligase complex, APC/C, adhesion molecules, integrins,Immunoglobulin-like adhesion molecules, selectins, cadherins, catenins,focal adhesion kinase, p130CAS, cytoskeletal/contractile proteins,fodrin, actin, paxillin, myosin, myosin binding proteins, tubulin,eg5/KSP, CENPs, heterotrimeric G proteins, β-adrenergic receptors,muscarinic receptors, adenylyl cyclase receptors, small molecular weightGTPases, H-Ras, K-Ras, N-Ras, Ran, Rac, Rho, Cdc42, Arfs, RABs, RHEB,guanine nucleotide exchange factors, Vav, Tiam, Sos, Dbl, PRK, TSC1,2,GTPase activating proteins, Ras-GAP, Arf-GAPs, Rho-GAPs, caspases,Caspase 2, Caspase 3, Caspase 6, Caspase 7, Caspase 8, Caspase 9, PARP,proteins involved in apoptosis, Bcl-2, Mcl-1, Bcl-XL, Bcl-w, Bcl-B, A1,Bax, Bak, Bok, Bik, Bad, Bid, Bim, Bmf, Hrk, Noxa, Puma, IAPB, XIAP,Smac, cell cycle regulators, Cdk4, Cdk 6, Cdk 2, Cdk1, Cdk 7, Cyclin D,Cyclin E, Cyclin A, Cyclin B, Rb, p16, p14Arf, p27KIP, p21CIP, molecularchaperones, Hsp90s, Hsp70, Hsp27, metabolic enzymes, Acetyl-CoAaCarboxylase, ATP citrate lyase, nitric oxide synthase, vesiculartransport proteins, caveolins, endosomal sorting complex required fortransport (ESCRT) proteins, vesicular protein sorting (Vsps),hydroxylases, prolyl-hydroxylases PHD-1, 2 and 3, asparagine hydroxylaseFIH transferases, isomerases, Pin1 prolyl isomerase, topoisomerases,deacetylases, Histone deacetylases, sirtuins, acetylases, histoneacetylases, CBP/P300 family, MYST family, ATF2, methylases, DNA methyltransferases, demethylases, Histone H3K4 demethylases, H3K27, JHDM2A,UTX, tumor suppressor genes, VHL, WT-1, p53, Hdm, PTEN, proteases,ubiquitin proteases, urokinase-type plasminogen activator (uPA) and uPAreceptor (uPAR) system, cathepsins, metalloproteinases, esterases,hydrolases, separase, ion channels, potassium channels, sodium channels,molecular transporters, multi-drug resistance proteins, P-Gycoprotein,nucleoside transporters, transcription factors/DNA binding proteins,Ets, Elk, SMADs, Rel-A (p65-NFB), CREB, NFAT, ATF-2, AFT, Myc, Fos, Sp1,Egr-1, T-bet, β-catenin, HIFs, FOXOs, E2Fs, SRFs, TCFs, Egr-1,β-□catenin, FOXO STAT1, STAT 3, STAT 4, STAT 5, STAT 6, p53, WT-1, HMGA,regulators of translation, pS6, 4EPB-1, eIF4E-binding protein,regulators of transcription, RNA polymerase, initiation factors,elongation factors. In some embodiments, the protein is S6.

In some embodiments, an epitope-recognizing fragment of an activationstate antibody rather than the whole antibody is used. In someembodiments, the epitope-recognizing fragment is immobilized. In someembodiments, the antibody light chain that recognizes an epitope isused. A recombinant nucleic acid encoding a light chain gene productthat recognizes an epitope may be used to produce such an antibodyfragment by recombinant means well known in the art.

Non-activation state antibodies may also be used in the presentinvention. In some embodiments, non-activation state antibodies bind toepitopes in both activated and non-activated forms of an element. Suchantibodies may be used to determine the amount of non-activated plusactivated element in a sample. In some embodiments, non-activation stateantibodies bind to epitopes present in non-activated forms of an elementbut absent in activated forms of an element. Such antibodies may be usedto determine the amount of non-activated element in a sample. Both typesof non-activation state antibodies may be used to determine if a changein the amount of activation state element, for example from samplesbefore and after treatment with a candidate bioactive agent as describedherein, coincide with changes in the amount of non-activation stateelement. For example, such antibodies can be used to determine whetheran increase in activated element is due to activation of non-activationstate element, or due to increased expression of the element, or both.

In some embodiments, antibodies are immobilized using beads analogous tothose known and used for standardization in flow cytometry. Attachmentof a multiplicity of activation state specific antibodies to beads maybe done by methods known in the art and/or described herein. Suchconjugated beads may be contacted with sample, preferably cell extract,under conditions that allow for a multiplicity of activated elements, ifpresent, to bind to the multiplicity of immobilized antibodies. A secondmultiplicity of antibodies comprising non-activation state antibodieswhich are uniquely labeled may be added to the immobilized activationstate specific antibody-activated element complex and the beads may besorted by FACS on the basis of the presence of each label, wherein thepresence of label indicates binding of corresponding second antibody andthe presence of corresponding activated element.

In alternative embodiments of the instant invention, aromatic aminoacids of protein binding elements may be replaced with D- orL-naphylalanine, D- or L-phenylglycine, D- or L-2-thieneylalanine, D- orL-1-, 2-, 3- or 4-pyreneylalanine, D- or L-3-thieneylalanine, D- orL-(2-pyridinyl)-alanine, D- or L-(3-pyridinyl)-alanine, D- orL-(2-pyrazinyl)-alanine, D- or L-(4-isopropyl)-phenylglycine,D-(trifluoromethyl)-phenylglycine, D-(trifluoromethyl)-phenylalanine,D-p-fluorophenylalanine, D- or L-p-biphenylphenylalanine, D- orL-p-methoxybiphenylphenylalanine, D- or L-2-indole(alkyl)alanines, andD- or L-alkylalanines where alkyl may be substituted or unsubstitutedmethyl, ethyl, propyl, hexyl, butyl, pentyl, isopropyl, iso-butyl,sec-isotyl, iso-pentyl, and non-acidic amino acids of C1-C20.

Acidic amino acids can be substituted with non-carboxylate amino acidswhile maintaining a negative charge, and derivatives or analogs thereof,such as the non-limiting examples of (phosphono)alanine, glycine,leucine, isoleucine, threonine, or serine; or sulfated (e.g., —SO3H)threonine, serine, or tyrosine.

Other substitutions may include non-natural hydroxylated amino acids maymade by combining “alkyl” with any natural amino acid. The term “alkyl”as used herein refers to a branched or unbranched saturated hydrocarbongroup of 1 to 24 carbon atoms, such as methyl, ethyl, n-propyl,isoptopyl, n-butyl, isobutyl, t-butyl, octyl, decyl, tetradecyl,hexadecyl, eicosyl, tetracisyl and the like. Alkyl includes heteroalkyl,with atoms of nitrogen, oxygen and sulfur. In some embodiments, alkylgroups herein contain 1 to 12 carbon atoms. Basic amino acids may besubstituted with alkyl groups at any position of the naturally occurringamino acids lysine, arginine, ornithine, citrulline, or(guanidino)-acetic acid, or other (guanidino)alkyl-acetic acids, where“alkyl” is define as above. Nitrile derivatives (e.g., containing theCN-moiety in place of COOH) may also be substituted for asparagine orglutamine, and methionine sulfoxide may be substituted for methionine.Methods of preparation of such peptide derivatives are well known to oneskilled in the art.

In addition, any amide linkage in any of the polypeptides may bereplaced by a ketomethylene moiety. Such derivatives are expected tohave the property of increased stability to degradation by enzymes, andtherefore possess advantages for the formulation of compounds which mayhave increased in vivo half lives, as administered by oral, intravenous,intramuscular, intraperitoneal, topical, rectal, intraocular, or otherroutes.

Additional amino acid modifications of amino acids of variantpolypeptides of to the present invention may include the following:Cysteinyl residues may be reacted with alpha-haloacetates (andcorresponding amines), such as 2-chloroacetic acid or chloroacetamide,to give carboxymethyl or carboxyamidomethyl derivatives. Cysteinylresidues may also be derivatized by reaction with compounds such asbromotrifluoroacetone, alpha-bromo-beta-(5-imidozoyl)propionic acid,chloroacetyl phosphate, N-alkylmaleimides, 3-nitro-2-pyridyl disulfide,methyl 2-pyridyl disulfide, p-chloromercuribenzoate,2-chloromercuri-4-nitrophenol, or chloro-7-nitrobenzo-2-oxa-1,3-diazole.

Histidyl residues may be derivatized by reaction with compounds such asdiethylprocarbonate e.g., at pH 5.5-7.0 because this agent is relativelyspecific for the histidyl side chain, and para-bromophenacyl bromide mayalso be used; e.g., where the reaction is preferably performed in 0.1Msodium cacodylate at pH 6.0.

Lysinyl and amino terminal residues may be reacted with compounds suchas succinic or other carboxylic acid anhydrides. Derivatization withthese agents is expected to have the effect of reversing the charge ofthe lysinyl residues.

Other suitable reagents for derivatizing alpha-amino-containing residuesinclude compounds such as imidoesters, e.g., as methyl picolinimidate;pyridoxal phosphate; pyridoxal; chloroborohydride;trinitrobenzenesulfonic acid; O-methylisourea; 2,4 pentanedione; andtransaminase-catalyzed reaction with glyoxylate. Arginyl residues may bemodified by reaction with one or several conventional reagents, amongthem phenylglyoxal, 2,3-butanedione, 1,2-cyclohexanedione, and ninhydrinaccording to known method steps. Derivatization of arginine residuesrequires that the reaction be performed in alkaline conditions becauseof the high pKa of the guanidine functional group. Furthermore, thesereagents may react with the groups of lysine as well as the arginineepsilon-amino group. The specific modification of tyrosyl residues perse is well known, such as for introducing spectral labels into tyrosylresidues by reaction with aromatic diazonium compounds ortetranitromethane.

N-acetylimidizol and tetranitromethane may be used to form O-acetyltyrosyl species and 3-nitro derivatives, respectively. Carboxyl sidegroups (aspartyl or glutamyl) may be selectively modified by reactionwith carbodiimides (R′—N—C—N—R′) such as1-cyclohexyl-3-(2-morpholiny-1-(4-ethyl) carbodiimide or1-ethyl-3-(4-azonia-4,4-dimethylpentyl) carbodiimide. Furthermoreaspartyl and glutamyl residues may be converted to asparaginyl andglutaminyl residues by reaction with ammonium ions.

Glutaminyl and asparaginyl residues may be frequently deamidated to thecorresponding glutamyl and aspartyl residues. Alternatively, theseresidues may be deamidated under mildly acidic conditions. Either formof these residues falls within the scope of the present invention.

In some embodiments, the activation state-specific binding element is apeptide comprising a recognition structure that binds to a targetstructure on an activatable protein. A variety of recognition structuresare well known in the art and can be made using methods known in theart, including by phage display libraries (see e.g., Gururaja et al.(2000) Chem. Biol. 7:515-27; Houimel et al., (2001) Eur. J. Immunol.31:3535-45; Cochran et al. (2001) J. Am. Chem. Soc. 123:625-32; Houimelet al. (2001) Int. J. Cancer 92:748-55, each incorporated herein byreference). Further, fluorophores can be attached to such antibodies foruse in the methods of the present invention.

A variety of recognitions structures are known in the art (e.g., Cochranet al., (2001) J. Am. Chem. Soc. 123:625-32; Boer et al., (2002) Blood100:467-73, each expressly incorporated herein by reference)) and can beproduced using methods known in the art (see e.g., Boer et al., (2002)Blood 100:467-73; Gualillo et al., (2002) Mol. Cell. Endocrinol.190:83-9, each expressly incorporated herein by reference)), includingfor example combinatorial chemistry methods for producing recognitionstructures such as polymers with affinity for a target structure on anactivatable protein (see e.g., Barn et al., (2001) J. Comb. Chem.3:534-41; Ju et al., (1999) Biotechnol. 64:232-9, each expresslyincorporated herein by reference). In another embodiment, the activationstate-specific antibody is a protein that only binds to an isoform of aspecific activatable protein that is phosphorylated and does not bind tothe isoform of this activatable protein when it is not phosphorylated ornon-phosphorylated. In another embodiment the activation state-specificantibody is a protein that only binds to an isoform of an activatableprotein that is intracellular and not extracellular, or vice versa. In asome embodiment, the recognition structure is an anti-lamininsingle-chain antibody fragment (scFv) (see e.g., Sanz et al., (2002)Gene Therapy 9:1049-53; Tse et al., (2002) J. Mol. Biol. 317:85-94, eachexpressly incorporated herein by reference).

In some embodiments the binding element is a nucleic acid. The term“nucleic acid” include nucleic acid analogs, for example, phosphoramide(Beaucage et al., (1993) Tetrahedron 49(10):1925 and references therein;Letsinger, J. (1970) Org. Chem. 35:3800; Sprinzl et al., (1977) Eur. J.Biochem. 81:579; Letsinger et al., (1986) Nucl. Acids Res. 14:3487;Sawai et al, (1984) Chem. Lett. 805, Letsinger et al., (1988) J. Am.Chem. Soc. 110:4470; and Pauwels et al., (1986) Chemica Scripta26:141-9), phosphorothioate (Mag et al., (1991) Nucleic Acids Res.19:1437; and U.S. Pat. No. 5,644,048), phosphorodithioate (Briu et al.,(1989) J. Am. Chem. Soc. 111:2321, O-methylphosphoroamidite linkages(see Eckstein, Oligonucleotides and Analogues: A Practical Approach,Oxford University Press), and peptide nucleic acid backbones andlinkages (see Egholm, (1992) J. Am. Chem. Soc. 114:1895; Meier et al.,(1992) Chem. Int. Ed. Engl. 31:1008; Nielsen, (1993) Nature, 365:566;Carlsson et al., (1996) Nature 380:207, all of which are incorporated byreference). Other analog nucleic acids include those with positivebackbones (Denpcy et al., (1995) Proc. Natl. Acad. Sci. USA 92:6097;non-ionic backbones (U.S. Pat. Nos. 5,386,023, 5,637,684, 5,602,240,5,216,141 and 4,469,863; Kiedrowshi et al., Angew. Chem. Intl. Ed.English 30:423 (1991); Letsinger et al., (1988) J. Am. Chem. Soc.110:4470; Letsinger et al., (1994) Nucleoside & Nucleotide 13:1597;Chapters 2 and 3, ASC Symposium Series 580, “Carbohydrate Modificationsin Antisense Research”, Ed. Y. S. Sanghui and P. Dan Cook; Mesmaeker etal., (1994) Bioorganic & Medicinal Chem. Lett. 4:395; Jeffs et al.,(1994) J. Biomolecular NMR 34:17; Tetrahedron Lett. 37:743 (1996)) andnon-ribose backbones, including those described in U.S. Pat. Nos.5,235,033 and 5,034,506, and Chapters 6 and 7, ASC Symposium Series 580,“Carbohydrate Modifications in Antisense Research”, Ed. Y. S. Sanghuiand P. Dan Cook. Nucleic acids containing one or more carbocyclic sugarsare also included within the definition of nucleic acids (see Jenkins etal., (1995) Chem. Soc. Rev. pp 169-176). Several nucleic acid analogsare described in Rawls, C & E News Jun. 2, 1997 page 35. All of thesereferences are hereby expressly incorporated by reference. Thesemodifications of the ribose-phosphate backbone may be done to facilitatethe addition of additional moieties such as labels, or to increase thestability and half-life of such molecules in physiological environments.

As will be appreciated by those in the art, all of these nucleic acidanalogs may find use in the present invention. In addition, mixtures ofnaturally occurring nucleic acids and analogs can be made.Alternatively, mixtures of different nucleic acid analogs, and mixturesof naturally occurring nucleic acids and analogs may be made. In someembodiments, peptide nucleic acids (PNA) which includes peptide nucleicacid analogs are used. These backbones are substantially non-ionic underneutral conditions, in contrast to the highly charged phosphodiesterbackbone of naturally occurring nucleic acids.

The nucleic acids may be single stranded or double stranded, asspecified, or contain portions of both double stranded or singlestranded sequence. The nucleic acid may be DNA, both genomic and cDNA,RNA or a hybrid, where the nucleic acid contains any combination ofdeoxyribo- and ribo-nucleotides, and any combination of bases, includinguracil, adenine, thymine, cytosine, guanine, inosine, xathaninehypoxathanine, isocytosine, isoguanine, etc.

In some embodiments, the binding element is a synthetic compound. Anynumbers of techniques are available for the random and directedsynthesis of a wide variety of organic compounds and biomolecules,including expression of randomized oligonucleotides. See for example WO94/24314, hereby expressly incorporated by reference, which discussesmethods for generating new compounds, including random chemistry methodsas well as enzymatic methods.

Alternatively, some embodiments utilize natural compounds, as bindingelements, in the form of bacterial, fungal, plant and animal extractsthat are available or readily produced.

Additionally, natural or synthetically produced compounds are readilymodified through conventional chemical, physical and biochemical means.Known pharmacological agents may be subjected to directed or randomchemical modifications, including enzymatic modifications, to producebinding elements that may be used in the instant invention.

In some embodiment the binding element is a small organic compound.Binding elements can be synthesized from a series of substrates that canbe chemically modified. “Chemically modified” herein includestraditional chemical reactions as well as enzymatic reactions. Thesesubstrates generally include, but are not limited to, alkyl groups(including alkanes, alkenes, alkynes and heteroalkyl), aryl groups(including arenes and heteroaryl), alcohols, ethers, amines, aldehydes,ketones, acids, esters, amides, cyclic compounds, heterocyclic compounds(including purines, pyrimidines, benzodiazepins, beta-lactams,tetracylines, cephalosporins, and carbohydrates), steroids (includingestrogens, androgens, cortisone, ecodysone, etc.), alkaloids (includingergots, vinca, curare, pyrollizdine, and mitomycines), organometalliccompounds, hetero-atom bearing compounds, amino acids, and nucleosides.Chemical (including enzymatic) reactions may be done on the moieties toform new substrates or binding elements that can then be used in thepresent invention.

In some embodiments the binding element is a carbohydrate. As usedherein the term carbohydrate is meant to include any compound with thegeneral formula (CH₂O)_(n). Examples of carbohydrates are di-, tri- andoligosaccharides, as well polysaccharides such as glycogen, cellulose,and starches.

In some embodiments the binding element is a lipid. As used herein theterm lipid herein is meant to include any water insoluble organicmolecule that is soluble in nonpolar organic solvents. Examples oflipids are steroids, such as cholesterol, and phospholipids such assphingomeylin.

Examples of activatable elements, activation states and methods ofdetermining the activation level of activatable elements are describedin US publication number 20060073474 entitled “Methods and compositionsfor detecting the activation state of multiple proteins in single cells”and US publication number 20050112700 entitled “Methods and compositionsfor risk stratification” the content of which are incorporate here byreference.

A. Labels

The methods and compositions of the instant invention provide bindingelements comprising a label or tag. By label is meant a molecule thatcan be directly (i.e., a primary label) or indirectly (i.e., a secondarylabel) detected; for example a label can be visualized and/or measuredor otherwise identified so that its presence or absence can be known. Acompound can be directly or indirectly conjugated to a label whichprovides a detectable signal, e.g. radioisotopes, fluorescers, enzymes,antibodies, particles such as magnetic particles, chemiluminescers, orspecific binding molecules, etc. Specific binding molecules includepairs, such as biotin and streptavidin, digoxin and antidigoxin etc.Examples of labels include, but are not limited to, optical fluorescentand chromogenic dyes including labels, label enzymes and radioisotopes.

In some embodiments, one or more binding elements are uniquely label.Using the example of two activation state specific antibodies, by“uniquely labeled” is meant that a first activation state antibodyrecognizing a first activated element comprises a first label, andsecond activation state antibody recognizing a second activated elementcomprises a second label, wherein the first and second labels aredetectable and distinguishable, making the first antibody and the secondantibody uniquely labeled.

In general, labels fall into four classes: a) isotopic labels, which maybe radioactive or heavy isotopes; b) magnetic, electrical, thermallabels; c) colored, optical labels including luminescent, phosphorousand fluorescent dyes or moieties; and d) binding partners. Labels canalso include enzymes (horseradish peroxidase, etc.) and magneticparticles. In some embodiments, the detection label is a primary label.A primary label is one that can be directly detected, such as afluorophore.

Labels include optical labels such as fluorescent dyes or moieties.Fluorophores can be either “small molecule” fluors, or proteinaceousfluors (e.g. green fluorescent proteins and all variants thereof).

Suitable fluorescent labels include, but are not limited to,fluorescein, rhodamine, tetramethylrhodamine, eosin, erythrosin,coumarin, methyl-coumarins, pyrene, Malacite green, stilbene, LuciferYellow, Cascade Blue™, Texas Red, IAEDANS, EDANS, BODIPY FL, LC Red 640,Cy 5, Cy 5.5, LC Red 705 and Oregon green. Suitable optical dyes aredescribed in the 1996 Molecular Probes Handbook by Richard P. Haugland,hereby expressly incorporated by reference. Suitable fluorescent labelsalso include, but are not limited to, green fluorescent protein (GFP;Chalfie, et al., Science 263(5148):802-805 (Feb. 11, 1994); and EGFP;Clontech—Genbank Accession Number U55762), blue fluorescent protein(BFP; 1. Quantum Biotechnologies, Inc. 1801 de Maisonneuve Blvd. West,8th Floor, Montreal (Quebec) Canada H3H 1J9; 2. Stauber, R. H.Biotechniques 24(3):462-471 (1998); 3. Heim, R. and Tsien, R. Y. Curr.Biol. 6:178-182 (1996)), enhanced yellow fluorescent protein (EYFP; 1.Clontech Laboratories, Inc., 1020 East Meadow Circle, Palo Alto, Calif.94303), luciferase (Ichiki, et al., J. Immunol. 150(12):5408-5417(1993)), .beta.-galactosidase (Nolan, et al., Proc Natl Acad Sci USA85(8):2603-2607 (April 1988)) and Renilla WO 92/15673; WO 95/07463; WO98/14605; WO 98/26277; WO 99/49019; U.S. Pat. No. 5,292,658; U.S. Pat.No. 5,418,155; U.S. Pat. No. 5,683,888; U.S. Pat. No. 5,741,668; U.S.Pat. No. 5,777,079; U.S. Pat. No. 5,804,387; U.S. Pat. No. 5,874,304;U.S. Pat. No. 5,876,995; and U.S. Pat. No. 5,925,558). All of theabove-cited references are expressly incorporated herein by reference.

In some embodiments, labels for use in the present invention include:Alexa-Fluor dyes (Alexa Fluor 350, Alexa Fluor 430, Alexa Fluor 488,Alexa Fluor 546, Alexa Fluor 568, Alexa Fluor 594, Alexa Fluor 633,Alexa Fluor 660, Alexa Fluor 680), Cascade Blue, Cascade Yellow andR-phycoerythrin (PE) (Molecular Probes) (Eugene, Oreg.), FITC,Rhodamine, and Texas Red (Pierce, Rockford, Ill.), Cy5, Cy5.5, Cy7(Amersham Life Science, Pittsburgh, Pa.). Tandem conjugate protocols forCy5PE, Cy5.5PE, Cy7PE, Cy5.5APC, Cy7APC are known in the art.Quantitation of fluorescent probe conjugation may be assessed todetermine degree of labeling and protocols including dye spectralproperties are also well known in the art. In some embodiments thefluorescent label is conjugated to an aminodextran linker which isconjugated to a binding element or antibody. Additional labels listed inand are available through the on-line and hard copy catalogues of BDBiosciences, Beckman Coulter, AnaSpec, Invitrogen, Cell SignalingTechnology, Millipore, eBioscience, Caltag, Santa Cruz Biotech, Abcamand Sigma, the contents of which are incorporated herein by reference.

In some embodiments, the fluorescent label is a GFP and, morepreferably, a Renilla, Ptilosarcus, or Aequorea species of GFP.

In some embodiments, a secondary detectable label is used. A secondarylabel is one that is indirectly detected; for example, a secondary labelcan bind or react with a primary label for detection, can act on anadditional product to generate a primary label (e.g. enzymes), etc.Secondary labels include, but are not limited to, one of a bindingpartner pair; chemically modifiable moieties; nuclease inhibitors,enzymes such as horseradish peroxidase, alkaline phosphatases,luciferases, etc.

In some embodiments, the secondary label is a binding partner pair. Forexample, the label may be a hapten or antigen, which will bind itsbinding partner. For example, suitable binding partner pairs include,but are not limited to: antigens (such as proteins (including peptides)and small molecules) and antibodies (including fragments thereof (FAbs,etc.)); proteins and small molecules, including biotin/streptavidin;enzymes and substrates or inhibitors; other protein-protein interactingpairs; receptor-ligands; and carbohydrates and their binding partners.Nucleic acid—nucleic acid binding proteins pairs are also useful.Binding partner pairs include, but are not limited to, biotin (orimino-biotin) and streptavidin, digeoxinin and Abs, and Prolinx™reagents.

In some embodiments, the binding partner pair comprises an antigen andan antibody that will specifically bind to the antigen. By “specificallybind” herein is meant that the partners bind with specificity sufficientto differentiate between the pair and other components or contaminantsof the system. The binding should be sufficient to remain bound underthe conditions of the assay, including wash steps to remove non-specificbinding. In some embodiments, the dissociation constants of the pairwill be less than about 10⁻⁴ to 10⁻⁹ M⁻¹, with less than about 10⁻⁵ to10⁻⁹ M⁻¹ being preferred and less than about 10⁻⁷ to 10⁻⁹ M⁻¹ beingparticularly preferred.

In some embodiment, the secondary label is a chemically modifiablemoiety. In this embodiment, labels comprising reactive functional groupsare incorporated into the molecule to be labeled. The functional groupcan then be subsequently labeled (e.g. either before or after the assay)with a primary label. Suitable functional groups include, but are notlimited to, amino groups, carboxy groups, maleimide groups, oxo groupsand thiol groups, with amino groups and thiol groups being particularlypreferred. For example, primary labels containing amino groups can beattached to secondary labels comprising amino groups, for example usinglinkers as are known in the art; for example, homo-orhetero-bifunctional linkers as are well known (see 1994 Pierce ChemicalCompany catalog, technical section on cross-linkers, pages 155-200,incorporated herein by reference).

In some embodiments, multiple fluorescent labels are employed in themethods and compositions of the present invention. In some embodiments,each lable is distinct and distinguishable from other labels.

As will be appreciated in the art antibody-label conjugation may beperformed using standard procedures or by usingprotein-protein/protein-dye cross-linking kits from Molecular Probes(Eugene, Oreg.).

In some embodiments, labeled antibodies are used for functional analysisof activatable proteins in cells. In performing such analysis severalareas of the experiment are considered: (1) identification of the propercombination of antibody cocktails for the stains (2), identification ofthe sequential procedure for the staining using the antigens (i.e., theactivatable protein) and antibody clones of interest, and (3) thoroughevaluation of cell culture conditions' effect on cell stimulation.Antigen clone selection is of particular importance for surface antigensof human cells, as different antibody clones yield different result anddo not stain similarly in different protocols. Selection of cell typesand optimization of culture conditions is also a critical component indetecting differences. For example, some cell lines have the ability toadapt to culture conditions and can yield heterogeneous responses.

In some embodiments, activation state-specific antibodies are labeledwith quantum dots as disclosed by Chattopadhyay, P. K. et al. Quantumdot semiconductor nanocrystals for immunophenotyping by polychromaticflow cytometry. Nat. Med. 12, 972-977 (2006). Quantum dot labels arecommercially available through Invitrogen,http://probes.invitrogen.com/products/qdot/.

Quantum dot labeled antibodies can be used alone or they can be employedin conjunction with organic fluorochrome-conjugated antibodies toincrease the total number of labels available. As the number of labeledantibodies increase so does the ability for subtyping known cellpopulations. Additionally, activation state-specific antibodies can belabeled using chelated or caged lanthanides as disclosed by Erkki, J. etal. Lanthanide chelates as new fluorochrome labels for cytochemistry. J.Histochemistry Cytochemistry, 36:1449-1451, 1988, and U.S. Pat. No.7,018,850, entitled Salicylamide-Lanthanide Complexes for Use asLuminescent Markers. Other methods of detecting fluorescence may also beused, e.g., Quantum dot methods (see, e.g., Goldman et al., J. Am. Chem.Soc. (2002) 124:6378-82; Pathak et al. J. Am. Chem. Soc. (2001)123:4103-4; and Remade et al., Proc. Natl. Sci. USA (2000) 18:553-8,each expressly incorporated herein by reference) as well as confocalmicroscopy.

In some embodiments, the activatable elements are labeled with tagssuitable for Inductively Coupled Plasma Mass Spectrometer (ICP-MS) asdisclosed in Tanner et al. Spectrochimica Acta Part B: AtomicSpectroscopy, 2007 March; 62(3):188-195; Ornatsky et al, mRNA Detectionin Leukemia Cell lines by Novel Metal-Tagged in situ Hybridization usingInductively Coupled Plasma Mass Spectometry, Translational Oncogenomics(2006):1, 1-9; Ornatsky et al, Multiple Cellular Antigen Detection byICP-MS, J. 1 mm. Methods 308 (2006) 68-76; and Lou et al., Polymer-BasedElemental Tags for Sensitive Bioassays, Angew. Chem. Int. Ed., (2007)46, 6111-6114.

Alternatively, detection systems based on FRET, discussed in detailbelow, may be used. FRET finds use in the instant invention, forexample, in detecting activation states that involve clustering ormultimerization wherein the proximity of two FRET labels is altered dueto activation. In some embodiments, at least two fluorescent labels areused which are members of a fluorescence resonance energy transfer(FRET) pair.

FRET is phenomenon known in the art wherein excitation of onefluorescent dye is transferred to another without emission of a photon.A FRET pair consists of a donor fluorophore and an acceptor fluorophore.The fluorescence emission spectrum of the donor and the fluorescenceabsorption spectrum of the acceptor must overlap, and the two moleculesmust be in close proximity. The distance between donor and acceptor atwhich 50% of donors are deactivated (transfer energy to the acceptor) isdefined by the Forster radius (Ro), which is typically 10-100 Å. Changesin the fluorescence emission spectrum comprising FRET pairs can bedetected, indicating changes in the number of that are in closeproximity (i.e., within 100 521 of each other). This will typicallyresult from the binding or dissociation of two molecules, one of whichis labeled with a FRET donor and the other of which is labeled with aFRET acceptor, wherein such binding brings the FRET pair in closeproximity. Binding of such molecules will result in an increasedfluorescence emission of the acceptor and/or quenching of thefluorescence emission of the donor.

FRET pairs (donor/acceptor) useful in the invention include, but are notlimited to, EDANS/fluorescein, IAEDANS/fluorescein,fluorescein/tetramethylrhodamine, fluorescein/LC Red 640, fluorescein/Cy5, fluorescein/Cy 5.5 and fluorescein/LC Red 705.

In some embodiments when FRET is used, a fluorescent donor molecule anda non-fluorescent acceptor molecule (“quencher”) may be employed. Inthis application, fluorescent emission of the donor will increase whenquencher is displaced from close proximity to the donor and fluorescentemission will decrease when the quencher is brought into close proximityto the donor. Useful quenchers include, but are not limited to, TAMRA,DABCYL, QSY 7 and QSY 33. Useful fluorescent donor/quencher pairsinclude, but are not limited to EDANS/DABCYL, Texas Red/DABCYL,BODIPY/DABCYL, Lucifer yellow/DABCYL, coumarin/DABCYL andfluorescein/QSY 7 dye.

The skilled artisan will appreciate that FRET and fluorescence quenchingallow for monitoring of binding of labeled molecules over time,providing continuous information regarding the time course of bindingreactions.

Preferably, changes in the degree of FRET are determined as a functionof the change in the ratio of the amount of fluorescence from the donorand acceptor moieties, a process referred to as “ratioing.” Changes inthe absolute amount of substrate, excitation intensity, and turbidity orother background absorbances in the sample at the excitation wavelengthaffect the intensities of fluorescence from both the donor and acceptorapproximately in parallel. Therefore the ratio of the two emissionintensities is a more robust and preferred measure of cleavage thaneither intensity alone.

The ratio-metric fluorescent reporter system described herein hassignificant advantages over existing reporters for protein integrationanalysis, as it allows sensitive detection and isolation of bothexpressing and non-expressing single living cells. In some embodiments,the assay system uses a non-toxic, non-polar fluorescent substrate thatis easily loaded and then trapped intracellularly. Modification of thefluorescent substrate by a cognate protein yields a fluorescent emissionshift as substrate is converted to product. Because the reporter readoutis ratiometric it is unique among reporter protein assays in that itcontrols for variables such as the amount of substrate loaded intoindividual cells. The stable, easily detected, intracellular readouteliminates the need for establishing clonal cell lines prior toexpression analysis. This system and other analogous flow sortingsystems can be used to isolate cells having a particular receptorelement clustering and/or activation profile from pools of millions ofviable cells.

The methods and composition of the present invention may also make useof label enzymes. By label enzyme is meant an enzyme that may be reactedin the presence of a label enzyme substrate that produces a detectableproduct. Suitable label enzymes for use in the present invention includebut are not limited to, horseradish peroxidase, alkaline phosphatase andglucose oxidase. Methods for the use of such substrates are well knownin the art. The presence of the label enzyme is generally revealedthrough the enzyme's catalysis of a reaction with a label enzymesubstrate, producing an identifiable product. Such products may beopaque, such as the reaction of horseradish peroxidase with tetramethylbenzedine, and may have a variety of colors. Other label enzymesubstrates, such as Luminol (available from Pierce Chemical Co.), havebeen developed that produce fluorescent reaction products. Methods foridentifying label enzymes with label enzyme substrates are well known inthe art and many commercial kits are available. Examples and methods forthe use of various label enzymes are described in Savage et al.,Previews 247:6-9 (1998), Young, J. Virol. Methods 24:227-236 (1989),which are each hereby incorporated by reference in their entirety.

By radioisotope is meant any radioactive molecule. Suitableradioisotopes for use in the invention include, but are not limited to¹⁴C, ³H, ³²P, ³³P, ³⁵S, ¹²⁵I, and ¹³¹I. The use of radioisotopes aslabels is well known in the art.

As mentioned, labels may be indirectly detected, that is, the tag is apartner of a binding pair. By “partner of a binding pair” is meant oneof a first and a second moiety, wherein the first and the second moietyhave a specific binding affinity for each other. Suitable binding pairsfor use in the invention include, but are not limited to,antigens/antibodies (for example, digoxigenin/anti-digoxigenin,dinitrophenyl (DNP)/anti-DNP, dansyl-X-anti-dansyl,Fluorescein/anti-fluorescein, lucifer yellow/anti-lucifer yellow, andrhodamine anti-rhodamine), biotin/avidin (or biotin/streptavidin) andcalmodulin binding protein (CBP)/calmodulin. Other suitable bindingpairs include polypeptides such as the FLAG-peptide [Hopp et al.,BioTechnology, 6:1204-1210 (1988)]; the KT3 epitope peptide [Martin etal., Science, 255: 192-194 (1992)]; tubulin epitope peptide [Skinner etal., J. Biol. Chem., 266:15163-15166 (1991)]; and the T7 gene 10 proteinpeptide tag [Lutz-Freyermuth et al., Proc. Natl. Acad. Sci. USA,87:6393-6397 (1990)] and the antibodies each thereto. As will beappreciated by those in the art, binding pair partners may be used inapplications other than for labeling, as is described herein.

As will be appreciated by those in the art, a partner of one bindingpair may also be a partner of another binding pair. For example, anantigen (first moiety) may bind to a first antibody (second moiety) thatmay, in turn, be an antigen for a second antibody (third moiety). Itwill be further appreciated that such a circumstance allows indirectbinding of a first moiety and a third moiety via an intermediary secondmoiety that is a binding pair partner to each.

As will be appreciated by those in the art, a partner of a binding pairmay comprise a label, as described above. It will further be appreciatedthat this allows for a tag to be indirectly labeled upon the binding ofa binding partner comprising a label. Attaching a label to a tag that isa partner of a binding pair, as just described, is referred to herein as“indirect labeling”.

By “surface substrate binding molecule” or “attachment tag” andgrammatical equivalents thereof is meant a molecule have bindingaffinity for a specific surface substrate, which substrate is generallya member of a binding pair applied, incorporated or otherwise attachedto a surface. Suitable surface substrate binding molecules and theirsurface substrates include, but are not limited to poly-histidine(poly-his) or poly-histidine-glycine (poly-his-gly) tags and Nickelsubstrate; the Glutathione-S Transferase tag and its antibody substrate(available from Pierce Chemical); the flu HA tag polypeptide and itsantibody 12CA5 substrate [Field et al., Mol. Cell. Biol., 8:2159-2165(1988)]; the c-myc tag and the 8F9, 3C7, 6E10, G4, B7 and 9E10 antibodysubstrates thereto [Evan et al., Molecular and Cellular Biology,5:3610-3616 (1985)]; and the Herpes Simplex virus glycoprotein D (gD)tag and its antibody substrate [Paborsky et al., Protein Engineering,3(6):547-553 (1990)]. In general, surface binding substrate moleculesuseful in the present invention include, but are not limited to,polyhistidine structures (His-tags) that bind nickel substrates,antigens that bind to surface substrates comprising antibody, haptensthat bind to avidin substrate (e.g., biotin) and CBP that binds tosurface substrate comprising calmodulin.

Production of antibody-embedded substrates is well known; see Slinkin etal., Bioconj. Chem., 2:342-348 (1991); Torchilin et al., supra;Trubetskoy et al., Bioconj. Chem. 3:323-327 (1992); King et al., CancerRes. 54:6176-6185 (1994); and Wilbur et al., Bioconjugate Chem.5:220-235 (1994) (all of which are hereby expressly incorporated byreference), and attachment of or production of proteins with antigens isdescribed above. Calmodulin-embedded substrates are commerciallyavailable, and production of proteins with CBP is described in Simcox etal., Strategies 8:40-43 (1995), which is hereby incorporated byreference in its entirety.

As will be appreciated by those in the art, tag-components of theinvention can be made in various ways, depending largely upon the formof the tag. Components of the invention and tags are preferably attachedby a covalent bond.

The production of tag-polypeptides by recombinant means when the tag isalso a polypeptide is described below. Production of tag-labeledproteins is well known in the art and kits for such production arecommercially available (for example, from Kodak and Sigma). Examples oftag labeled proteins include, but are not limited to, a Flag-polypeptideand His-polypeptide. Methods for the production and use of tag-labeledproteins are found, for example, in Winston et al., Genes and Devel.13:270-283 (1999), incorporated herein in its entirety, as well asproduct handbooks provided with the above-mentioned kits.

Biotinylation of target molecules and substrates is well known, forexample, a large number of biotinylation agents are known, includingamine-reactive and thiol-reactive agents, for the biotinylation ofproteins, nucleic acids, carbohydrates, carboxylic acids; see chapter 4,Molecular Probes Catalog, Haugland, 6th Ed. 1996, hereby incorporated byreference. A biotinylated substrate can be attached to a biotinylatedcomponent via avidin or streptavidin. Similarly, a large number ofhaptenylation reagents are also known (Id.).

Methods for labeling of proteins with radioisotopes are known in theart. For example, such methods are found in Ohta et al., (1999) Molec.Cell 3:535-541, which is hereby incorporated by reference in itsentirety.

Production of proteins having tags by recombinant means is well known,and kits for producing such proteins are commercially available. Forexample, such a kit and its use are described in the QIAexpress Handbookfrom Qiagen by Joanne Crowe et al., hereby expressly incorporated byreference.

The functionalization of labels with chemically reactive groups such asthiols, amines, carboxyls, etc. is generally known in the art. In someembodiments, the tag is functionalized to facilitate covalentattachment. The covalent attachment of the tag may be either direct orvia a linker. In one embodiment, the linker is a relatively shortcoupling moiety, which is used to attach the molecules. A couplingmoiety may be synthesized directly onto a component of the invention andcontains at least one functional group to facilitate attachment of thetag. Alternatively, the coupling moiety may have at least two functionalgroups, which are used to attach a functionalized component to afunctionalized tag, for example. In an additional embodiment, the linkeris a polymer. In this embodiment, covalent attachment is accomplishedeither directly, or through the use of coupling moieties from thecomponent or tag to the polymer. In some embodiments, the covalentattachment is direct, that is, no linker is used. In this embodiment,the component preferably contains a functional group such as acarboxylic acid that is used for direct attachment to the functionalizedtag. It should be understood that the component and tag may be attachedin a variety of ways, including those listed above. In some embodiments,the tag is attached to the amino or carboxy terminus of the polypeptide.As will be appreciated by those in the art, the above description of thecovalent attachment of a label applies to the attachment of virtuallyany two molecules of the present disclosure.

In some embodiments, the tag is functionalized to facilitate covalentattachment, as is generally outlined above. Thus, a wide variety of tagsare commercially available which contain functional groups, including,but not limited to, isothiocyanate groups, amino groups, haloacetylgroups, maleimides, succinimidyl esters, and sulfonyl halides, all ofwhich may be used to covalently attach the tag to a second molecule, asis described herein. The choice of the functional group of the tag willdepend on the site of attachment to either a linker, as outlined aboveor a component of the invention. Thus, for example, for direct linkageto a carboxylic acid group of a protein, amino modified or hydrazinemodified tags will be used for coupling via carbodiimide chemistry, forexample using 1-ethyl-3-(3-dimethylaminopropyl)-carbodiimi-de (EDAC) asis known in the art (see Set 9 and Set 11 of the Molecular ProbesCatalog, supra; see also the Pierce 1994 Catalog and Handbook, pagesT-155 to T-200, both of which are hereby incorporated by reference). Inone embodiment, the carbodiimide is first attached to the tag, such asis commercially available for many of the tags described herein.

Alternative Activation State Indicators

An alternative activation state indicator useful with the instantinvention is one that allows for the detection of activation byindicating the result of such activation. For example, phosphorylationof a substrate can be used to detect the activation of the kinaseresponsible for phosphorylating that substrate. Similarly, cleavage of asubstrate can be used as an indicator of the activation of a proteaseresponsible for such cleavage. Methods are well known in the art thatallow coupling of such indications to detectable signals, such as thelabels and tags described above in connection with binding elements. Forexample, cleavage of a substrate can result in the removal of aquenching moiety and thus allowing for a detectable signal beingproduced from a previously quenched label.

Modulators

In some embodiments, the methods and composition utilize a modulator. Amodulator can be an activator, an inhibitor or a compound capable ofimpacting a cellular pathway. Modulators can take the form ofenvironmental cues and inputs.

Modulation can be performed in a variety of environments. In someembodiments, cells are exposed to a modulator immediately aftercollection. In some embodiments where there is a mixed population ofcells, purification of cells is performed after modulation. In someembodiments, whole blood is collected to which a modulator is added. Insome embodiments, cells are modulated after processing for single cellsor purified fractions of single cells. As an illustrative example, wholeblood can be collected and processed for an enriched fraction oflymphocytes that is then exposed to a modulator. Modulation can includeexposing cells to more than one modulator. For instance, in someembodiments, cells are exposed to at least 2, 3, 4, 5, 6, 7, 8, 9, or 10modulators.

In some embodiments, cells are cultured post collection in a suitablemedia before exposure to a modulator. In some embodiments, the media isa growth media. In some embodiments, the growth media is a complex mediathat may include serum. In some embodiments, the growth media comprisesserum. In some embodiments, the serum is selected from the groupconsisting of fetal bovine serum, bovine serum, human serum, porcineserum, horse serum, and goat serum. In some embodiments, the serum levelranges from 0.0001% to 30%. In some embodiments any suitable amount ofserum is used. In some embodiments, the growth media is a chemicallydefined minimal media and is without serum. In some embodiments, cellsare cultured in a differentiating media.

Modulators include chemical and biological entities, and physical orenvironmental stimuli. Modulators can act extracellularly orintracellularly. Chemical and biological modulators include growthfactors, cytokines, neurotransmitters, adhesion molecules, hormones,small molecules, inorganic compounds, polynucleotides, antibodies,natural compounds, lectins, lactones, chemotherapeutic agents,biological response modifiers, carbohydrate, proteases and freeradicals. Modulators include complex and undefined biologic compositionsthat may comprise cellular or botanical extracts, cellular or glandularsecretions, physiologic fluids such as serum, amniotic fluid, or venom.Physical and environmental stimuli include electromagnetic, ultraviolet,infrared or particulate radiation, redox potential and pH, the presenceor absences of nutrients, changes in temperature, changes in oxygenpartial pressure, changes in ion concentrations and the application ofoxidative stress. Modulators can be endogenous or exogenous and mayproduce different effects depending on the concentration and duration ofexposure to the single cells or whether they are used in combination orsequentially with other modulators. Modulators can act directly on theactivatable elements or indirectly through the interaction with one ormore intermediary biomolecule. Indirect modulation includes alterationsof gene expression wherein the expressed gene product is the activatableelement or is a modulator of the activatable element.

In some embodiments, modulators produce different activation statesdepending on the concentration of the modulator, duration of exposure orwhether they are used in combination or sequentially with othermodulators.

In some embodiments the modulator is selected from the group consistingof growth factor, cytokine, adhesion molecule modulator, drugs, hormone,small molecule, polynucleotide, antibodies, natural compounds, lactones,chemotherapeutic agents, immune modulator, carbohydrate, proteases,ions, reactive oxygen species, peptides, and protein fragments, eitheralone or in the context of cells, cells themselves, viruses, andbiological and non-biological complexes (e.g. beads, plates, viralenvelopes, antigen presentation molecules such as majorhistocompatibility complex). In some embodiments, the modulator is aphysical stimuli such as heat, cold, UV radiation, and radiation.Examples of modulators, include but are not limited to, F(ab)2 IgM,Rituxan, Alemtuzumab, anti CD22 (epratuzumab), anti-CD23 (lumiliximab),Campath, H₂O₂, PMA, BAFF, April, SDFla, CD40L, IGF-1, Imiquimod,polyCpG, fludarabine, cyclophosphamide, chlorambucil, IL-7, IL-6, IL-10,IL-27, IL-4, IL-2, IL-3, and thapsigargin.

In some embodiments, the modulator is an activator. In some embodimentsthe modulator is an inhibitor. In some embodiments, cells are exposed toone or more modulator. In some embodiments, cells are exposed to atleast 2, 3, 4, 5, 6, 7, 8, 9, or 10 modulators. In some embodiments,cells are exposed to at least two modulators, wherein one modulator isan activator and one modulator is an inhibitor. In some embodiments,cells are exposed to at least 2, 3, 4, 5, 6, 7, 8, 9, or 10 modulators,where at least one of the modulators is an inhibitor.

In some embodiments, the modulator is a B cell receptor modulator. Insome embodiments, the B cell receptor modulator is a B cell receptoractivator. An example of B cell receptor activator is a cross-linker ofthe B cell receptor complex or the B-cell co-receptor complex. In someembodiments, cross-linker is an antibody or molecular binding entity. Insome embodiments, the cross-linker is an antibody. In some embodiments,the antibody is a multivalent antibody. In some embodiments, theantibody is a monovalent, bivalent, or multivalent antibody made moremultivalent by attachment to a solid surface or tethered on ananoparticle surface to increase the local valency of the epitopebinding domain.

In some embodiments, the cross-linker is a molecular binding entity. Insome embodiments, the molecular binding entity acts upon or binds the Bcell receptor complex via carbohydrates or an epitope in the complex. Insome embodiments, the molecular is a monovalent, bivalent, ormultivalent is made more multivalent by attachment to a solid surface ortethered on a nanoparticle surface to increase the local valency of theepitope binding domain.

In some embodiments, the cross-linking of the B cell receptor complex orthe B-cell co-receptor complex comprises binding of an antibody ormolecular binding entity to the cell and then causing its crosslinkingvia interaction of the cell with a solid surface that causescrosslinking of the BCR complex via antibody or molecular bindingentity.

In some embodiments, the crosslinker is F(ab)2 IgM, IgG, IgD, polyclonalBCR antibodies, monoclonal BCR antibodies, Fc receptor derived bindingelements and/or a combination thereof. The Ig can be derived from aspecies selected from the group consisting of mouse, goat, rabbit, pig,rat, horse, cow, shark, chicken, or llama. In some embodiments, thecrosslinker is F(ab)2 IgM, Polyclonal IgM antibodies, Monoclonal IgMantibodies, Biotinylated F(ab)2 IgCM, Biotinylated Polyclonal IgMantibodies, Biotinylated Monoclonal IgM antibodies and/or combinationthereof.

In some embodiments, the inhibitor is an inhibitor of a cellular factoror a plurality of factors that participates in a cellular pathway (e.g.signaling cascade) in the cell. In some embodiments, the inhibitor is akinase or phosphatase inhibitor. Examples of kinase inhibitors includeadaphostin, AG 490, AG 825, AG 957, AG 1024, aloisine, aloisine A,alsterpaullone, aminogenistein, API-2, apigenin, arctigenin, AY-22989,BAY 61-3606, bisindolylmaleimide IX, chelerythrine,10-[4′-(N,N-Diethylamino)butyl]-2-chlorophenoxazine hydrochloride,dasatinib, 2-Dimethylamino-4,5,6,7-tetrabromo-1H-benzimidazole,5,7-Dimethoxy-3-(4-pyridinyl)quinoline dihydrochloride, edelfosine,ellagic acid, enzastaurin, ER 27319 maleate, erlotinib, ET18OCH3,fasudil, flavopiridol, gefitinib, GW 5074, H-7, H-8, H-89, HA-100,HA-1004, HA-1077, HA-1100, hydroxyfasudil, indirubin-3′-oxime,5-Iodotubercidin, kenpaullone, KN-62, KY12420, LFM-A13, lavendustin A,luteolin, LY-294002, LY294002, mallotoxin, ML-9, NSC-154020, NSC-226080,NSC-231634, NSC-664704, NSC-680410, NU6102, olomoucine, oxindole I,PD-153035, PD-98059, PD 169316, phloretin, phloridzin, piceatannol,picropodophyllin, PK1, PP1, PP2, purvalanol A, quercetin, R406, R788,rapamune, rapamycin, Ro 31-8220, roscovitine, rottlerin, SB202190,SB203580, sirolimus, sorafenib, SL327, SP600125, staurosporine, STI-571,SU-11274, SU1498, SU4312, SU6656, 4,5,6,7-Tetrabromotriazole, TG101348,Triciribine, Tyrphostin AG 490, Tyrphostin AG 825, Tyrphostin AG 957,Tyrphostin AG 1024, Tyrphostin SU1498, U0126, VX-509, VX-667, VX-680,W-7, wortmannin, XL-019, XL-147, XL-184, XL-228, XL-281, XL-518, XL-647,XL-765, XL-820, XL-844, XL-880, Y-27632, ZD-1839, ZM-252868, ZM-447439,siRNA, miRNA Examples of phosphatase inhibitors include, but are notlimited to H₂O₂, siRNA, miRNA, Cantharidin, (−)-p-Bromotetramisole,Microcystin LR, Sodium Orthovanadate, Sodium Pervanadate, Vanadylsulfate, Sodium oxodiperoxo(1,10-phenanthroline)vanadate,bis(maltolato)oxovanadium(IV), Sodium Molybdate, Sodium Perm olybdate,Sodium Tartrate, Imidazole, Sodium Fluoride, β-Glycerophosphate, SodiumPyrophosphate Decahydrate, Calyculin A, Discodermia calyx, bpV(phen),mpV(pic), DMHV, Cypermethrin, Dephostatin, Okadaic Acid, NIPP-1,N-(9,10-Dioxo-9,10-dihydro-phenanthren-2-yl)-2,2-dimethyl-propionamide,α-Bromo-4-hydroxyacetophenone, 4-Hydroxyphenacyl Br,α-Bromo-4-methoxyacetophenone, 4-Methoxyphenacyl Br,α-Bromo-4-(carboxymethoxy)acetophenone, 4-(Carboxymethoxy)phenacyl Br,and bis(4-Trifluoromethylsulfonamidophenyl)-1,4-diisopropylbenzene,phenyarsine oxide, Pyrrolidine Dithiocarbamate, and Aluminum fluoride.In some embodiments, the phosphatase inhibitor is H₂O₂.

In some embodiments H₂O₂ is administered as an inhibitor. In someembodiments H₂O₂ is administered at between 0.01 and 50 mM. In someembodiments H₂O₂ is administered at between 0.1 and 10 mM. In someembodiments H₂O₂ is administered at between 1 and 10 mM. In someembodiments H₂O₂ is administered at between 1 and 5 mM. In someembodiments H₂O₂ is administered at 0.5, 1, 1.5, 2, 2.5, 3, 3.5, 4, 4.5,5, 5.5, 6, 6.5, 7, 7.5, 8, 8.5, 9, 9.5 or 10 mM. In certain embodiments,H₂O₂ is administered at 3.0 mM. In certain embodiments, H₂O₂ isadministered at 3.3 mM. In some embodiments the duration of exposure ofH₂O₂ is between 0.01 and 360 minutes. In some embodiments the durationof exposure of H₂O₂ is between 0.1 and 240 minutes. In some embodimentsthe duration of exposure of H₂O₂ is between 0.5 and 180 minutes. In someembodiments the duration of exposure of H₂O₂ is between 0 and 120minutes. In some embodiments the duration of exposure to H₂O₂ is between5 and 15 minutes. In some embodiments the duration of exposure of H₂O₂is 1, 2, 3, 4, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 70, 80,90, 100, 110, 120, 140, 160 or 180 minutes. In some embodiments theduration of exposure of H₂O₂ is 10 minutes. In some embodiments H₂O₂ isadministered as an inhibitor with at least one other modulator. In someembodiments H₂O₂ is administered as an inhibitor with F(ab)2 IgM or anysuitable BCR agonist. In some embodiments H₂O₂ is administered beforeadministration of F(ab)2 IgM. In some embodiments H₂O₂ is administeredsimultaneously with F(ab)2 IgM. In some embodiments H₂O₂ is administeredafter F(ab)2 IgM.

In some embodiments, the activation level of an activatable element in acell is determined after contacting the cell with at least 2, 3, 4, 5,6, 7, 8, 9, or 10 modulators. In some embodiments, the activation levelof an activatable element in a cell is determined after contacting thecell with at least 2, 3, 4, 5, 6, 7, 8, 9, or 10 modulators where atleast one of the modulators is an inhibitor. In some embodiments, theactivation level of an activatable element in a cell is determined aftercontacting the cell with an inhibitor and a modulator, where themodulator can be an inhibitor or an activator. In some embodiments, theactivation level of an activatable element in a cell is determined aftercontacting the cell with an inhibitor and an activator. In someembodiments, the activation level of an activatable element in a cell isdetermined after contacting the cell with two or more modulators.

In some embodiments, a phenotypic profile of a population of cells isdetermined by measuring the activation level of an activatable elementwhen the population of cells is exposed to a plurality of modulators inseparate cultures. In some embodiments, the modulators include F(ab)2IgM, Rituxan, Alemtuzumab, anti CD22 (epratuzumab), anti-CD23(lumiliximab), Campath, H₂O₂, PMA, BAFF, April, SDF1a, CD40L, IGF-1,Imiquimod, polyCpG, fludarabine, cyclophosphamide, chlorambucil, IL-7,IL-6, IL-10, IL-27, IL-4, IL-2, IL-3, thapsigardin and/or a combinationthereof. For instance a population of cells can be exposed to one ormore, all or a combination of the following combination of modulators:(i) F(ab)2 IgM; (ii) Rituxan,; (iii) Campath; (iv) H₂O₂; (v) PMA; (vi)BAFF; (vii) April; (viii) SDF1a; (ix) CD40L; (x) IGF-1; (xi) Imiquimod;(xii) polyCpG; (xiii) fludarabine; (xiv) cyclophosphamide;(xv)chlorambucil; IL-7; (xvi) IL-6; (xvii)IL-10; (xviii) IL-27; (xx) IL-4;(xx) IL-2; (xxi) IL-3; (xxii) Alemtuzumab, (xxiii) anti CD22(epratuzumab), (xxiv) anti-CD23 (lumiliximab), (xxv) thapsigargin; and(xxvi) F(ab)2 IgM and H₂O₂. In some embodiments, the phenotypic profileof the population of cells is used to classify the population asdescribed herein.

Detection

In practicing the methods of this invention, the detection of the statusof the one or more activatable elements can be carried out by a person,such as a technician in the laboratory. Alternatively, the detection ofthe status of the one or more activatable elements can be carried outusing automated systems. In either case, the detection of the status ofthe one or more activatable elements for use according to the methods ofthis invention is performed according to standard techniques andprotocols well-established in the art.

One or more activatable elements can be detected and/or quantified byany method that detect and/or quantitates the presence of theactivatable element of interest. Such methods may includeradioimmunoassay (RIA) or enzyme linked immunoabsorbance assay (ELISA),immunohistochemistry, immunofluorescent histochemistry with or withoutconfocal microscopy, reversed phase assays, homogeneous enzymeimmunoassays, and related non-enzymatic techniques, Western blots, wholecell staining, immunoelectronmicroscopy, nucleic acid amplification,gene array, protein array, mass spectrometry, patch clamp, 2-dimensionalgel electrophoresis, differential display gel electrophoresis,microsphere-based multiplex protein assays, label-free cellular assaysand flow cytometry, etc. U.S. Pat. No. 4,568,649 describes liganddetection systems, which employ scintillation counting. These techniquesare particularly useful for modified protein parameters. Cell readoutsfor proteins and other cell determinants can be obtained usingfluorescent or otherwise tagged reporter molecules. Flow cytometrymethods are useful for measuring intracellular parameters.

In some embodiments, the present invention provides methods fordetermining an activatable element's activation profile for a singlecell. The methods may comprise analyzing cells by flow cytometry on thebasis of the activation level of at least two activatable elements.Binding elements (e.g. activation state-specific antibodies) are used toanalyze cells on the basis of activatable element activation level, andcan be detected as described below. Alternatively, non-binding elementssystems as described above can be used in any system described herein.

When using fluorescent labeled components in the methods andcompositions of the present invention, it will recognized that differenttypes of fluorescent monitoring systems, e.g., Cytometric measurementdevice systems, can be used to practice the invention. In someembodiments, flow cytometric systems are used or systems dedicated tohigh throughput screening, e.g. 96 well or greater microtiter plates.Methods of performing assays on fluorescent materials are well known inthe art and are described in, e.g., Lakowicz, J. R., Principles ofFluorescence Spectroscopy, New York: Plenum Press (1983); Herman, B.,Resonance energy transfer microscopy, in: Fluorescence Microscopy ofLiving Cells in Culture, Part B, Methods in Cell Biology, vol. 30, ed.Taylor, D. L. & Wang, Y.-L., San Diego: Academic Press (1989), pp.219-243; Turro, N.J., Modern Molecular Photochemistry, Menlo Park:Benjamin/Cummings Publishing Col, Inc. (1978), pp. 296-361.

Fluorescence in a sample can be measured using a fluorimeter. Ingeneral, excitation radiation, from an excitation source having a firstwavelength, passes through excitation optics. The excitation opticscause the excitation radiation to excite the sample. In response,fluorescent proteins in the sample emit radiation that has a wavelengththat is different from the excitation wavelength. Collection optics thencollect the emission from the sample. The device can include atemperature controller to maintain the sample at a specific temperaturewhile it is being scanned. According to one embodiment, a multi-axistranslation stage moves a microtiter plate holding a plurality ofsamples in order to position different wells to be exposed. Themulti-axis translation stage, temperature controller, auto-focusingfeature, and electronics associated with imaging and data collection canbe managed by an appropriately programmed digital computer. The computeralso can transform the data collected during the assay into anotherformat for presentation. In general, known robotic systems andcomponents can be used.

Other methods of detecting fluorescence may also be used, e.g., Quantumdot methods (see, e.g., Goldman et al., J. Am. Chem. Soc. (2002)124:6378-82; Pathak et al. J. Am. Chem. Soc. (2001) 123:4103-4; andRemade et al., Proc. Natl. Sci. USA (2000) 18:553-8, each expresslyincorporated herein by reference) as well as confocal microscopy. Ingeneral, flow cytometry involves the passage of individual cells throughthe path of a laser beam. The scattering the beam and excitation of anyfluorescent molecules attached to, or found within, the cell is detectedby photomultiplier tubes to create a readable output, e.g. size,granularity, or fluorescent intensity.

The detecting, sorting, or isolating step of the methods of the presentinvention can entail fluorescence-activated cell sorting (FACS)techniques, where FACS is used to select cells from the populationcontaining a particular surface marker, or the selection step can entailthe use of magnetically responsive particles as retrievable supports fortarget cell capture and/or background removal. A variety of FACS systemsare known in the art and can be used in the methods of the invention(see e.g., WO99/54494, filed Apr. 16, 1999; U.S. Ser. No. 20010006787,filed Jul. 5, 2001, each expressly incorporated herein by reference).

In some embodiments, a FACS cell sorter (e.g. a FACSVantage™ CellSorter, Becton Dickinson Immunocytometry Systems, San Jose, Calif.) isused to sort and collect cells based on their activation profile(positive cells) in the presence or absence of a change in activationlevel in an activatable element in response to a modulator. In someembodiments the change is a decrease. In some embodiments the change isan increase.

In some embodiments, the cells are first contacted withfluorescent-labeled activation state-specific binding elements (e.g.antibodies) directed against specific activation state of specificactivatable elements. In such an embodiment, the amount of bound bindingelement on each cell can be measured by passing droplets containing thecells through the cell sorter. By imparting an electromagnetic charge todroplets containing the positive cells, the cells can be separated fromother cells. The positively selected cells can then be harvested insterile collection vessels. These cell-sorting procedures are describedin detail, for example, in the FACSVantage™. Training Manual, withparticular reference to sections 3-11 to 3-28 and 10-1 to 10-17, whichis hereby incorporated by reference in its entirety.

In another embodiment, positive cells can be sorted using magneticseparation of cells based on the presence of an isoform of anactivatable element. In such separation techniques, cells to bepositively selected are first contacted with specific binding element(e.g., an antibody or reagent that binds an isoform of an activatableelement). The cells are then contacted with retrievable particles (e.g.,magnetically responsive particles) that are coupled with a reagent thatbinds the specific element. The cell-binding element-particle complexcan then be physically separated from non-positive or non-labeled cells,for example, using a magnetic field. When using magnetically responsiveparticles, the positive or labeled cells can be retained in a containerusing a magnetic filed while the negative cells are removed. These andsimilar separation procedures are described, for example, in the BaxterImmunotherapy Isolex training manual which is hereby incorporated in itsentirety.

In some embodiments, methods for the determination of a receptor elementactivation state profile for a single cell are provided. The methodscomprise providing a population of cells and analyze the population ofcells by flow cytometry. Preferably, cells are analyzed on the basis ofthe activation level of at least two activatable elements. In someembodiments, a multiplicity of activatable element activation-stateantibodies is used to simultaneously determine the activation level of amultiplicity of elements.

In some embodiment, cell analysis by flow cytometry on the basis of theactivation level of at least two elements is combined with adetermination of other flow cytometry readable outputs, such as thepresence of surface markers, granularity and cell size to provide acorrelation between the activation level of a multiplicity of elementsand other cell qualities measurable by flow cytometry for single cells.

As will be appreciated, the present invention also provides for theordering of element clustering events in signal transduction.Particularly, the present invention allows the artisan to construct anelement clustering and activation hierarchy based on the correlation oflevels of clustering and activation of a multiplicity of elements withinsingle cells. Ordering can be accomplished by comparing the activationlevel of a cell or cell population with a control at a single timepoint, or by comparing cells at multiple time points to observesubpopulations arising out of the others.

The present invention provides a valuable method of determining thepresence of cellular subsets within cellular populations. Ideally,signal transduction pathways are evaluated in homogeneous cellpopulations to ensure that variances in signaling between cells do notqualitatively nor quantitatively mask signal transduction events andalterations therein. As the ultimate homogeneous system is the singlecell, the present invention allows the individual evaluation of cells toallow true differences to be identified in a significant way.

Thus, the invention provides methods of distinguishing cellular subsetswithin a larger cellular population. As outlined herein, these cellularsubsets often exhibit altered biological characteristics (e.g.activation levels, altered response to modulators) as compared to othersubsets within the population. For example, as outlined herein, themethods of the invention allow the identification of subsets of cellsfrom a population such as primary cell populations, e.g. peripheralblood mononuclear cells that exhibit altered responses (e.g. responseassociated with presence of a condition) as compared to other subsets.In addition, this type of evaluation distinguishes between differentactivation states, altered responses to modulators, cell lineages, celldifferentiation states, etc.

As will be appreciated, these methods provide for the identification ofdistinct signaling cascades for both artificial and stimulatoryconditions in complex cell populations, such a peripheral bloodmononuclear cells, or naive and memory lymphocytes.

When necessary, cells are dispersed into a single cell suspension (e.g.by enzymatic digestion with a suitable protease, collagenase, dispase,etc; and the like). An appropriate solution is used for dispersion orsuspension. Such solution will generally be a balanced salt solution,e.g. normal saline, PBS, Hanks balanced salt solution, etc.,conveniently supplemented with fetal calf serum or other naturallyoccurring factors, in conjunction with an acceptable buffer at lowconcentration, generally from 5-25 mM. Convenient buffers include HEPES1phosphate buffers, lactate buffers, etc. The cells may be fixed, e.g.with 3% paraformaldehyde, and are usually permeabilized, e.g. with icecold methanol; HEPES-buffered PBS containing 0.1% saponin, 3% BSA;covering for 2 min in acetone at −200 C; and the like as known in theart and according to the methods described herein.

In some embodiments, one or more cells are contained in a well of a 96well plate or other commercially available multi-well plate. In analternate embodiment, the reaction mixture or cells are in a cytometricmeasurement device. Other multi-well plates useful in the presentinvention include, but are not limited to 384 well plates and 1536 wellplates. Still other vessels for containing the reaction mixture or cellsand useful in the present invention will be apparent to the skilledartisan.

The addition of the components of the assay for detecting the activationlevel or activity of an activatable element, or modulation of suchactivation level or activity, may be sequential or in a predeterminedorder or grouping under conditions appropriate for the activity that isassayed for. Such conditions are described here and known in the art.Moreover, further guidance is provided below (see, e.g., in theExamples).

In some embodiments, the activation level of an activatable element ismeasured using Inductively Coupled Plasma Mass Spectrometer (ICP-MS). Abinding element that has been labeled with a specific element binds tothe activatable element. When the cell is introduced into the ICP, it isatomized and ionized. The elemental composition of the cell, includingthe labeled binding element that is bound to the activatable element, ismeasured. The presence and intensity of the signals corresponding to thelabels on the binding element indicates the level of the activatableelement on that cell (Tanner et al. Spectrochimica Acta Part B: AtomicSpectroscopy, (2007), 62(3):188-195.).

As will be appreciated by one of skill in the art, the instant methodsand compositions find use in a variety of other assay formats inaddition to flow cytometry analysis. For example, a chip analogous to aDNA chip can be used in the methods of the present invention. Arrayersand methods for spotting nucleic acid to a chip in a prefigured arrayare known. In addition, protein chips and methods for synthesis areknown. These methods and materials may be adapted for the purpose ofaffixing activation state binding elements to a chip in a prefiguredarray. In some embodiments, such a chip comprises a multiplicity ofelement activation state binding elements, and is used to determine anelement activation state profile for elements present on the surface ofa cell.

In some embodiments, a chip comprises a multiplicity of the “second setbinding elements,” in this case generally unlabeled. Such a chip iscontacted with sample, preferably cell extract, and a secondmultiplicity of binding elements comprising element activation statespecific binding elements is used in the sandwich assay tosimultaneously determine the presence of a multiplicity of activatedelements in sample. Preferably, each of the multiplicity of activationstate-specific binding elements is uniquely labeled to facilitatedetection.

In some embodiments confocal microscopy can be used to detect activationprofiles for individual cells. Confocal microscopy relies on the serialcollection of light from spatially filtered individual specimen points,which is then electronically processed to render a magnified image ofthe specimen. The signal processing involved confocal microscopy has theadditional capability of detecting labeled binding elements withinsingle cells, accordingly in this embodiment the cells can be labeledwith one or more binding elements. In some embodiments the bindingelements used in connection with confocal microscopy are antibodiesconjugated to fluorescent labels, however other binding elements, suchas other proteins or nucleic acids are also possible.

In some embodiments, the methods and compositions of the instantinvention can be used in conjunction with an “In-Cell Western Assay.” Insuch an assay, cells are initially grown in standard tissue cultureflasks using standard tissue culture techniques. Once grown to optimumconfluency, the growth media is removed and cells are washed andtrypsinized. The cells can then be counted and volumes sufficient totransfer the appropriate number of cells are aliquoted into microwellplates (e.g., Nunc™ 96 Microwell™ plates). The individual wells are thengrown to optimum confluency in complete media whereupon the media isreplaced with serum-free media. At this point controls are untouched,but experimental wells are incubated with a modulator, e.g. EGF. Afterincubation with the modulator cells are fixed and stained with labeledantibodies to the activation elements being investigated. Once the cellsare labeled, the plates can be scanned using an imager such as theOdyssey Imager (LiCor, Lincoln Nebr.) using techniques described in theOdyssey Operator's Manual v1.2., which is hereby incorporated in itsentirety. Data obtained by scanning of the multi-well plate can beanalyzed and activation profiles determined as described below.

In some embodiments, the detecting is by high pressure liquidchromatography (HPLC), for example, reverse phase HPLC, and in a furtheraspect, the detecting is by mass spectrometry.

These instruments can fit in a sterile laminar flow or fume hood, or areenclosed, self-contained systems, for cell culture growth andtransformation in multi-well plates or tubes and for hazardousoperations. The living cells may be grown under controlled growthconditions, with controls for temperature, humidity, and gas for timeseries of the live cell assays. Automated transformation of cells andautomated colony pickers may facilitate rapid screening of desiredcells.

Flow cytometry or capillary electrophoresis formats can be used forindividual capture of magnetic and other beads, particles, cells, andorganisms.

Flexible hardware and software allow instrument adaptability formultiple applications. The software program modules allow creation,modification, and running of methods. The system diagnostic modulesallow instrument alignment, correct connections, and motor operations.Customized tools, labware, and liquid, particle, cell and organismtransfer patterns allow different applications to be performed.Databases allow method and parameter storage. Robotic and computerinterfaces allow communication between instruments.

In some embodiment, the methods of the invention include the use ofliquid handling components. The liquid handling systems can includerobotic systems comprising any number of components. In addition, any orall of the steps outlined herein may be automated; thus, for example,the systems may be completely or partially automated.

As will be appreciated by those in the art, there are a wide variety ofcomponents which can be used, including, but not limited to, one or morerobotic arms; plate handlers for the positioning of microplates;automated lid or cap handlers to remove and replace lids for wells onnon-cross contamination plates; tip assemblies for sample distributionwith disposable tips; washable tip assemblies for sample distribution;96 well loading blocks; cooled reagent racks; microtiter plate pipettepositions (optionally cooled); stacking towers for plates and tips; andcomputer systems.

Fully robotic or microfluidic systems include automated liquid-,particle-, cell- and organism-handling including high throughputpipetting to perform all steps of screening applications. This includesliquid, particle, cell, and organism manipulations such as aspiration,dispensing, mixing, diluting, washing, accurate volumetric transfers;retrieving, and discarding of pipet tips; and repetitive pipetting ofidentical volumes for multiple deliveries from a single sampleaspiration. These manipulations are cross-contamination-free liquid,particle, cell, and organism transfers. This instrument performsautomated replication of microplate samples to filters, membranes,and/or daughter plates, high-density transfers, full-plate serialdilutions, and high capacity operation. Additional examples ofautomation, automated sample collection and analysis are disclosed inU.S. 61/048,657 which is hereby incorporated by reference in itsentirety.

In some embodiments, chemically derivatized particles, plates,cartridges, tubes, magnetic particles, or other solid phase matrix withspecificity to the assay components are used. The binding surfaces ofmicroplates, tubes or any solid phase matrices include non-polarsurfaces, highly polar surfaces, modified dextran coating to promotecovalent binding, antibody coating, affinity media to bind fusionproteins or peptides, surface-fixed proteins such as recombinant proteinA or G, nucleotide resins or coatings, and other affinity matrix areuseful in this invention.

In some embodiments, platforms for multi-well plates, multi-tubes,holders, cartridges, minitubes, deep-well plates, microfuge tubes,cryovials, square well plates, filters, chips, optic fibers, beads, andother solid-phase matrices or platform with various volumes areaccommodated on an upgradeable modular platform for additional capacity.This modular platform includes a variable speed orbital shaker, andmulti-position work decks for source samples, sample and reagentdilution, assay plates, sample and reagent reservoirs, pipette tips, andan active wash station. In some embodiments, the methods of theinvention include the use of a plate reader.

In some embodiments, thermocycler and thermoregulating systems are usedfor stabilizing the temperature of heat exchangers such as controlledblocks or platforms to provide accurate temperature control ofincubating samples from 0° C. to 100° C.

In some embodiments, interchangeable pipet heads (single ormulti-channel) with single or multiple magnetic probes, affinity probes,or pipetters robotically manipulate the liquid, particles, cells, andorganisms. Multi-well or multi-tube magnetic separators or platformsmanipulate liquid, particles, cells, and organisms in single or multiplesample formats.

In some embodiments, the instrumentation will include a detector, whichcan be a wide variety of different detectors, depending on the labelsand assay. In some embodiments, useful detectors include a microscope(s)with multiple channels of fluorescence; plate readers to providefluorescent, ultraviolet and visible spectrophotometric detection withsingle and dual wavelength endpoint and kinetics capability,fluorescence resonance energy transfer (FRET), luminescence, quenching,two-photon excitation, and intensity redistribution; CCD cameras tocapture and transform data and images into quantifiable formats; and acomputer workstation.

In some embodiments, the robotic apparatus includes a central processingunit which communicates with a memory and a set of input/output devices(e.g., keyboard, mouse, monitor, printer, etc.) through a bus. Again, asoutlined below, this may be in addition to or in place of the CPU forthe multiplexing devices of the invention. The general interactionbetween a central processing unit, a memory, input/output devices, and abus is known in the art. Thus, a variety of different procedures,depending on the experiments to be run, are stored in the CPU memory.

These robotic fluid handling systems can utilize any number of differentreagents, including buffers, reagents, samples, washes, assay componentssuch as label probes, etc.

Analysis

Advances in flow cytometry have enabled the individual cell enumerationof up to thirteen simultaneous parameters (De Rosa et al., 2001) and aremoving towards the study of genomic and proteomic data subsets (Krutzikand Nolan, 2003; Perez and Nolan, 2002). Likewise, advances in othertechniques (e.g. microarrays) allow for the identification of multipleactivatable elements. As the number of parameters, epitopes, and sampleshave increased, the complexity of experiments and the challenges of dataanalysis have grown rapidly. An additional layer of data complexity hasbeen added by the development of stimulation panels which enable thestudy of activatable elements under a growing set of experimentalconditions. Methods for the analysis of multiple parameters are wellknown in the art. In some embodiments flow cytometry applicationsrequire software for different phases of operation and analysis, see61/079,579; 61/079,551; 61/079,537; 61/087,555; 61/085,789 which arehereby incorporated by reference in their entireties.

In some embodiments where flow cytometry is used, flow cytometryexperiments are arrayed and the results are approximated as fold changesusing a heat map to facilitate evaluation. Generally speaking, arrayedflow cytometry experiments simplify multidimensional flow cytometry databased on experimental design and observed differences between flowcytometry samples. One common way of comparing changes in a set of flowcytometry samples is to overlay histograms of one parameter on the sameplot. Arrayed flow cytometry experiments ideally contain a referencesample against which experimental samples are compared. This referencesample is placed in the first position of the array, and subsequentexperimental samples follow the control in the sequence. Referencesamples can include normal and/or cells associated with a condition(e.g. tumor cells).

In some embodiments where flow cytometry is used, prior to analyzing ofdata the populations of interest and the method for characterizing thesepopulations are determined. For instance, there are at least two generalways of identifying populations for data analysis: (i) “Outside-in”comparison of Parameter sets for individual samples or subset (e.g.,patients in a trial). In this more common case, cell populations arehomogenous or lineage gated in such a way as to create distinct setsconsidered to be homogenous for targets of interest. An example ofsample-level comparison would be the identification of signalingprofiles in tumor cells of a patient and correlation of these profileswith non-random distribution of clinical responses. This is consideredan outside-in approach because the population of interest is pre-definedprior to the mapping and comparison of its profile to other populations.(ii) “Inside-out” comparison of Parameters at the level of individualcells in a heterogeneous population. An example of this would be thesignal transduction state mapping of mixed hematopoietic cells undercertain conditions and subsequent comparison of computationallyidentified cell clusters with lineage specific markers. This could beconsidered an inside-out approach to single cell studies as it does notpresume the existence of specific populations prior to classification. Amajor drawback of this approach is that it creates populations which, atleast initially, require multiple transient markers to enumerate and maynever be accessible with a single cell surface epitope. As a result, thebiological significance of such populations can be difficult todetermine. The main advantage of this unconventional approach is theunbiased tracking of cell populations without drawing potentiallyarbitrary distinctions between lineages or cell types.

Each of these techniques capitalizes on the ability of flow cytometry todeliver large amounts of multiparameter data at the single cell level.For cells associated with a condition (e.g. neoplastic or hematopoeticcondition), a third “meta-level” of data exists because cells associatedwith a condition (e.g. cancer cells) are generally treated as a singleentity and classified according to historical techniques. Thesetechniques have included organ or tissue of origin, degree ofdifferentiation, proliferation index, metastatic spread, and genetic ormetabolic data regarding the patient.

In some embodiments, the present invention uses variance mappingtechniques for mapping condition signaling space. These methodsrepresent a significant advance in the study of condition biologybecause it enables comparison of conditions independent of a putativenormal control. Traditional differential state analysis methods (e.g.,DNA microarrays, subtractive Northern blotting) generally rely on thecomparison of cells associated with a condition from each patient samplewith a normal control, generally adjacent and theoreticallyuntransformed tissue. Alternatively, they rely on multiple clusteringsand re-clusterings to group and then further stratify patient samplesaccording to phenotype. In contrast, variance mapping of conditionstates compares condition samples first with themselves and then againstthe parent condition population. As a result, activation states with themost diversity among conditions provide the core parameters in thedifferential state analysis. Given a pool of diverse conditions, thistechnique allows a researcher to identify the molecular events thatunderlie differential condition pathology (e.g., cancer responses tochemotherapy), as opposed to differences between conditions and aproposed normal control.

In some embodiments, when variance mapping is used to profile thesignaling space of patient samples, conditions whose signaling responseto modulators is similar are grouped together, regardless of tissue orcell type of origin. Similarly, two conditions (e.g. two tumors) thatare thought to be relatively alike based on lineage markers or tissue oforigin could have vastly different abilities to interpret environmentalstimuli and would be profiled in two different groups.

When groups of signaling profiles have been identified it is frequentlyuseful to determine whether other factors, such as clinical responses,presence of gene mutations, and protein expression levels, arenon-randomly distributed within the groups. If experiments or literaturesuggest such a hypothesis in an arrayed flow cytometry experiment, itcan be judged with simple statistical tests, such as the Student'st-test and the X² test. Similarly, if two variable factors within theexperiment are thought to be related, the r² correlation coefficientfrom a linear regression is used to represent the degree of thisrelationship.

Examples of analysis for activatable elements are described in USpublication number 20060073474 entitled “Methods and compositions fordetecting the activation state of multiple proteins in single cells” andUS publication number 20050112700 entitled “Methods and compositions forrisk stratification” and U.S. 61/085,789 the contents of which areincorporate here by reference.

CLL serves as an example of the methods of the invention. The data shownin FIGS. 26, 27 and 28 is a heat map comparing the activation states ofmultiple activatable elements in 22 CLL patients and 4 control patients.This data demonstrates that B-cells from various CLL patients displaydistinguishable patterns of activatable elements as visualized by a heatmap. An inhibitor or inhibitor plus another modulator further defineadditional patterns of activatable elements that allow identification,classification and grouping of cryptic or aberrant hematopoeticpopulations (i.e. patient clustering). In FIGS. 26, 27 & 28 patientsamples are indicated at the top of the heat map. Each column representsa single patient. CLL indicates that the sample was obtained from apatient diagnosed with CLL. CON indicates that the sample was obtainedfrom a control patient. The heat map legend is indicated at the top ofthe figure and uses a shaded scale based on the log10-fold increase, ordecrease, in mean fluorescence intensity (MFI), relative to theunstimulated control (0 min).

The heat map defines the activation state of various activatableelements by denoting a change, or lack thereof, in the level of anactivatable element revealed by the presence of an inhibitor and/oradditional modulator. Thus, the heat map can define the presence orabsence of an increase in the activation level of a plurality ofactivatable elements in a cell upon contacting said cell with aninhibitor or a modulator. Labels to the right of the heat map indicatethe activatable element detected, e.g. a phospho-protein. Labels to theright also indicate the modulator or inhibitor treatment for that row.“US” indicates unstimulated or untreated. FIG. 28 illustrates a patternof activation levels of a plurality of activatable elements in a cell.FIG. 28 further illustrates the identification of patient clusteringgroups (i.e. clustering groups). A patient clustering group is comprisedof samples from patients that display similar or distinct patterns ofactivation levels in one or more activatable elements in response to oneor more modulators (e.g., an inhibitor, or an inhibitor and anothermodulator). FIG. 28 illustrates a clustering group comprised of samplesfrom patients in which the activation levels of p-PLCγ₂, p-SyK/Zap-70,p-BLNK and p-Lck are similar in response to the same stimulus. Somepatient clustering groups are revealed upon modulation or treatment withan inhibitor as illustrated by the boxed regions. Treatment with H₂O₂reveals a patient clustering group defined by the levels of p-PLCγ₂,p-SyK/Zap-70, p-BLNK and p-Lck (FIG. 28, bottom right boxed area) thatare similar to those of the four control patients (FIG. 28, bottomcenter box). Treatment with H₂O₂ further reveals a patient clusteringgroup that is distinct from the controls (FIG. 28, 9 patients to theleft of bottom boxed area). Modulation with H₂O₂ and BCR crosslinkingdefines another patient clustering group comprised of samples frompatients that display the activation levels of p-BLNK, p-Syk and p-PLCγ₂(FIG. 28, top left boxed area) that are similar to the control patients(top center box). In addition, modulation with H₂O₂ and BCR crosslinkingfurther reveals another clustering group distinct from the controls (10patients to the right of top boxed area).

Thus, also provided herein is a method of deriving a classification.Deriving a classification involves defining a clustering group. Aclustering group is defined by determining the activation state of aplurality of activatable elements from a plurality of cells wherein eachcell is derived from an individual with a known conditions and/or knownclinical outcome. A clustering group may define a pattern thatassociated with a known condition or known clinical outcome. Anysuitable activatable element can be used wherein the activation level ofsaid activatable element provides useful information regarding a knowncondition or clinical outcome of a patient. A cell derived from apatient with an unknown condition and/or unknown clinical outcome may beclassified depending upon which clustering group it is identified with.This can further lead to diagnosis, prognosis, and/or evaluation orchoice of treatment for the patient.

Kits

In some embodiments the invention provides kits. Kits provided by theinvention may comprise one or more of the state-specific binding elementdescribed herein, such as phospho-specific antibodies. In someembodiments, the kit comprises one or more of the phospho-specificantibodies specific for the proteins selected from the group consistingof PI3-Kinase (p85, p110a, p110b, p110d), Jak1, Jak2, SOCs, Rac, Rho,Cdc42, Ras-GAP, Vav, Tiam, Sos, Dbl, Nck, Gab, PRK, SHPT, and SHP2,SHIP1, SHIP2, sSHIP, PTEN, Shc, Grb2, PDK1, SGK, Akt1, Akt2, Akt3,TSC1,2, Rheb, mTor, 4EBP-1, p70S6Kinase, S6, LKB-1, AMPK, PFK,Acetyl-CoAa Carboxylase, DokS, Rafs, Mos, Tp12, MEK1/2, MLK3, TAK, DLK,MKK3/6, MEKK1,4, MLK3, ASK1, MKK4/7, SAPK/JNK1,2,3, p38s, Erk1/2, Syk,Btk, BLNK, LAT, ZAP70, Lck, Cbl, SLP-76, PLCγ₁, PLCγ₂, STAT1, STAT 3,STAT 4, STAT 5, STAT 6, FAK, p130CAS, PAKs, LIMK1/2, Hsp90, Hsp70,Hsp27, SMADs, Rel-A (p65-NFKB), CREB, Histone H2B, HATs, HDACs, PKR, Rb,Cyclin D, Cyclin E, Cyclin A, Cyclin B, P16, p14Arf, p27KIP, p21CIP,Cdk4, Cdk6, Cdk7, Cdk1, Cdk2, Cdk9, Cdc25,A/B/C, Abl, E2F, FADD, TRADD,TRAF2, RIP, Myd88, BAD, Bcl-2, Mcl-1, Bcl-XL, Caspase 2, Caspase 3,Caspase 6, Caspase 7, Caspase 8, Caspase 9, PARP, IAPB, Smac, Fodrin,Actin, Src, Lyn, Fyn, Lck, NIK, IKB, p65(RelA), IKKα, PKA, PKCα, PKCβ,PKCθ, PKCδ, CAMK, Elk, AFT, Myc, Egr-1, NFAT, ATF-2, Mdm2, p53, DNA-PK,Chk1, Chk2, ATM, ATR, β-catenin, CrkL, GSK3α, GSK3β, and FOXO. In someembodiments, the kit comprises one or more of the phospho-specificantibodies specific for the proteins selected from the group consistingof Erk, Syk, Zap70, Lck, Btk, BLNK, Cbl, PLCγ₂, Akt, RelA, p38, S6. Insome embodiments, the kit comprises one or more of the phospho-specificantibodies specific for the proteins selected from the group consistingof Akt1, Akt2, Akt3, SAPK/JNK1,2,3, p38s, Erk1/2, Syk, ZAP70, Btk, BLNK,Lck, PLCγ, PLC1γ₂, STAT1, STAT 3, STAT 4, STAT 5, STAT 6, CREB, Lyn,p-S6, Cbl, NF-κB, GSK3β, CARMA/Bcl10 and Tcl-1.

Kits provided by the invention may comprise one or more of themodulators described herein. In some embodiments, the kit comprises oneor more modulators selected from the group consisting of F(ab)2 IgM,H₂O₂, PMA, BAFF, April, SDF1a, CD40L, IGF-1, Imiquimod, polyCpG, IL-7,IL-6, IL-10, IL-27, IL-4, IL-2, IL-3, thapsigargin and a combinationthereof.

The state-specific binding element of the invention can be conjugated toa solid support and to detectable groups directly or indirectly. Thereagents may also include ancillary agents such as buffering agents andstabilizing agents, e.g., polysaccharides and the like. The kit mayfurther include, where necessary, other members of the signal-producingsystem of which system the detectable group is a member (e.g., enzymesubstrates), agents for reducing background interference in a test,control reagents, apparatus for conducting a test, and the like. The kitmay be packaged in any suitable manner, typically with all elements in asingle container along with a sheet of printed instructions for carryingout the test.

Such kits enable the detection of activatable elements by sensitivecellular assay methods, such as IHC and flow cytometry, which aresuitable for the clinical detection, prognosis, and screening of cellsand tissue from patients, such as leukemia patients, having a diseaseinvolving altered pathway signaling.

Such kits may additionally comprise one or more therapeutic agents. Thekit may further comprise a software package for data analysis of thephysiological status, which may include reference profiles forcomparison with the test profile.

Such kits may also include information, such as scientific literaturereferences, package insert materials, clinical trial results, and/orsummaries of these and the like, which indicate or establish theactivities and/or advantages of the composition, and/or which describedosing, administration, side effects, drug interactions, or otherinformation useful to the health care provider. Such kits may alsoinclude instructions to access a database such as described in U.S. Ser.No. 61/087,555 for selecting an antibody specific for the pathway ofinterest. Such information may be based on the results of variousstudies, for example, studies using experimental animals involving invivo models and studies based on human clinical trials. Kits describedherein can be provided, marketed and/or promoted to health providers,including physicians, nurses, pharmacists, formulary officials, and thelike. Kits may also, in some embodiments, be marketed directly to theconsumer.

The following examples serve to more fully describe the manner of usingthe above-described invention, as well as to set forth the best modescontemplated for carrying out various aspects of the invention. It isunderstood that these examples in no way serve to limit the true scopeof this invention, but rather are presented for illustrative purposes.All references cited herein are expressly incorporated by reference intheir entirety.

EXAMPLES Example 1 Signaling Pathways in CLL Samples

Signals propagated through the B cell receptor (BCR) guide thematuration and survival of B cells and might factor in the pathogenesisand progression of chronic lymphocytic leukemia (CLL). In this example,BCR signaling in CLL cells was investigated at the single-cell levelusing multiparametric flow cytometry. Concurrent analysis was performedusing fluorochrome-conjugated antibodies specific for B-cell surfaceantigens and a panel of antibodies recognizing specific phospho-peptideepitopes within a selected group of intracellular signaling proteins.CLL samples from patients (N=6) showed weak or minimal signalingactivity at p72SYK/p70ZAP, Erk1/2, B-Cell linker protein (BLNK) andphospholipase-Cγ-2, (PLCγ₂) when stimulated only at the BCR with anti-μcrosslinking, whereas a robust signal was observed in a control Ramos Bcell line. The low-level signaling in CLL cells could be accounted forby either a defect in activation of a key protein required forsignaling, or by enhanced inhibition mediated by phosphatases such asSHIP-1, SHIP-2, SHP-1, or SHP-2. To determine whether phosphatases werepreventing or dampening BCR activation in CLL samples, CLL cells weretreated with hydrogen peroxide (H₂O₂), a physiologic phosphataseinhibitor generated during BCR signaling that has been used previouslyto reveal dysregulated BCR signaling in follicular lymphoma (Sing etal., (2005) Cell; Reth (2002) Nat. Immunol.; Irish et al., (2006) Blood.H₂O₂ treatment of CLL cells induced high-levels of phosphorylatedp72SYK/p70ZAP, ERK1/2, BLNK, and PLCγ₂, in some patients independent ofsurface F(ab)2anti-μ ligation. In contrast, other CLL-B cells weresignificantly less responsive to this treatment, even in the presence ofF(ab)2anti-μ. Exposure of blood B cells from healthy donors to H₂O₂failed to elicit a substantial increase in phosphorylation of these sameintracellular signaling proteins. These studies reveal a previouslyunrecognized, constitutive high-level phosphatase activity in some CLLcells possibly contributing to the attenuated signaling observed inthese cells following surface IgM ligation.

Analysis of Signaling Pathways in CLL Cells

Ramos cells were maintained and cultured using methods known in the art.Cells from CLL patients were obtained using methods known in the art.

BCR cross-linking and preparation for staining: Cells were thawed at 37°C. in a water bath until partially thawed (˜50%). Cells were thawed into5% FBS/RPMI at room temperature. The cell suspension was added drop-wiseinto media. Cells were centrifuged at 900 RPM for 10 minutes with nobrake. The supernatants were decanted and pellets resuspended in 25milliliters (ml) of media. Cells were re-centrifuged at 900 RPM for 10minutes, decanted again and resuspended in 5 ml of 1% FBS/RPMI. Cellswere counted with a hemocytometer using trypan blue staining. Theconcentration of the cells was adjusted as necessary to reduce crowding.Cells were incubated at 37° C. for 2 hours. The final cellconcentration, including additives and reagents (e.g. stimulant, Fabfragment, H₂O₂, aqua, etc.) was 1.6 million/ml. Cells were aliquotedinto wells. F(ab)2IgM alone, 3.3 mM H₂O₂ alone or F(ab)2IgM and H₂O₂(H₂O₂ added within 30 seconds of F(ab)2IgM) were added to theappropriate tubes and mixed by vortexing the samples. Cells wereincubated with the different treatments for 15 minutes, unless a timecourse was performed. If a time course was performed, the cells wereincubated with the different treatments for 5, 10, 30, 60, or 120minutes. After incubation, the cells were fixed with 1.6%paraformaldehyde for 5 minutes at room temperature in the dark. Cellswere then washed with 0.1% BSA/PBS (2 ml) and centrifuged at 2000 RPMfor 5 minutes. The supernatant were decanted and pellet resuspended in 1ml of 100% MeOH (methanol).

Staining: Tubes were washed with 2 ml of 0.4% BSA PBS (ice cold) andcentrifuged at 2000 RPM for 10 minutes at 4° C. Cells were washed twice.Then cells were stained with fluorescent conjugated antibodies specificfor CD20, CD3, CD4, CD8, CD5, p-Erk, p-BLNK, p-syk/Zap70, for 25 min. atRT. Cells were placed into 80 wells making a total of 2 plates. Thecocktail mix used for the staining is described below:

-   -   a. Cocktail        -   (i) CD3 pac blue=160 μl        -   (ii) CD4 APC=80 μl        -   (iii) CD8 PE Cy7=80 μl        -   (iv) CD5 PE Cy5=400 μl        -   (v) CD20 PerCP Cy5.5=800 μl        -   (vi) p-Erk=800 μl        -   (vii) p-BLNK=800 μl        -   (viii) p-Syk/ZAP70=800 μl    -   b. Total antibody volume in the cocktail mix was 3920 μl. PBS        was added to bring the total volume to 8.0 ml. Each well        received 100 μl of this cocktail mix.

Analysis by Flow Cytometry: Between 10,000 and 100,000 un-gated eventswere collected for each sample on the BD LSR II flow cytometry machine.The fluorescent anti-bodies directed to extra-cellular markers (i.e.CD3, CD4, CD8, CD5 and CD20) were used to mark the cells and ahierarchical gating strategy was used to identify the B-Cells and matureB-Cell population among the recorded events as described below:

-   -   a. Forward and side scatter were first used to gate the        Lymphocyte (Lymph) population.    -   b. This “Lymph” population was then displayed on the CD20 and        CD3 axes to gate for high CD3 expressing cells (CD3+) and high        CD20 expressing cells (CD20+).    -   c. The “CD20+” population was further gated to identify the        mature B-Cell (high CD5 expressing cells—CD5+) population by        displaying them on the CD20 and CD5 axes.    -   d. All the recorded events were also marked by the following        intra-cellular markers p-Erk, p-Syk/Zap70, and p-BLNK.

A probability density estimate of log₁₀ of the fluorescence intensityvalue of each of the intra-cellular markers was computed for the cellsin the CD20+ and CD5+ using the kernel density estimation function onthe R-Statistics package (http://www.r-project.org/). The probabilitydensity estimates provided were then plotted to visualize the change inthe phopho-levels of the various markers from the base-line.

Results: FIG. 1 shows that both F(ab)₂ IgM and PMA activates p-Erk andp-Syk/pZap70 in Ramos cells. The large dashed line on the histogramsrepresent the level of fluorescence of unstimulated and unstained cellsreferred herein after as autofluorescence. The thick solid linerepresents the level of fluorescence of unstimulated and stained cellsreferred hereinafter as background. After both stimulation with PMA(dotted line) and F(ab)₂ IgM (thin solid line) the p-Erk and p-Sykfluorescence is above the autofluorescence and background signalindicating activation of those proteins upon stimulation. FIG. 2 showsthat F(ab)₂ IgM (dotted line) also activates pBLNK, pCbl, pPLCγ₂, pLck,p38 in Ramos cells. Ramos cells (Ramos cell lines) are used as positivecontrol throughout the experiments performed herein.

Cells from CLL patients were analyzed. FIG. 3 shows that PMA (thin solidline) activates Erk in pheresed CLL samples. However, increasing amountsof amounts of F(ab)₂IgM for 15 min minimally activated signaling in CLLsamples (FIG. 4).

CLL samples were divided into populations with low and high frequency ofZAP70. Zap70 can be a prognostic indicator used for CLL. However theclinical implications of CLL samples that contain ZAP70 are unclear. CLLsamples show modest p-Erk (y-axis) and p-Syk (x-axis) phosphorylationwhen activated with PMA (FIG. 5). These cells show weak activation whenactivated with F(ab)₂IgM as shown in FIG. 5.

CLL samples were treated with H₂O₂ or in combination with F(ab)₂IgM.H₂O₂ is a known inhibitor of phosphatases. Inhibition of phosphatasesactivates Erk and Syk as shown in FIG. 6. Without intending to belimited to any theory, inhibition of phosphatases reveals strong tonicBCR signaling. This tonic signaling is not apparent by F(ab)₂IgM alone(FIG. 4-5). F(ab)₂IgM and H₂O₂ reveals different kinetics subpopulation(heterogeneity) differences between CLL patients (FIG. 6). In contrast,in normal B cells H₂O₂ blocks phosphatases, but is incapable ofactivating post BCR by itself (FIG. 7). Without meaning to be limited toany theory, these results suggest that these CLLs have alterations insignaling proximal to the BCR. In CLL H₂O₂ reveals an underlyingsignaling event, possibly a tonic signaling, that can drive signalingevents downstream of BCR (such as Syk and Erk) as shown in FIG. 8.

Other CLL samples show modest and weak Erk and Syk phosphorylation whenactivated with PMA and F(ab)₂IgM, respectively (FIG. 9). When these CLLsamples are treated with H₂O₂, inhibition of phosphatases does notreveal strong tonic BCR signaling (FIG. 10). Treatment with F(ab)₂IgMand H₂O₂ reveals: (i) different kinetics (ii) subpopulationheterogeneity and (iii) differences between CLL patients as shown inFIG. 10.

Kinetics of Signaling in CLL Specimens

Cells were prepared and stained as described above.

FIG. 12 shows different kinetics of Syk and Erk phosphorylation byF(ab)₂IgM and H₂O₂. Peak phosphorylation of Syk and BLNK occurred after5 min of activation, whereas peak phosphorylation of Erk after 30 min.

FIG. 13 shows moderate activation of Syk/Zap70 and Erk by F(ab)₂IgM andH₂O₂. FIG. 14 shows the kinetics of signaling by F(ab)₂IgM and H₂O₂.FIG. 21 shows minimal activation of Syk/Zap70 and Erk by F(ab)2IgM aloneover time. FIG. 22 shows minimal activation of Syk/Zap70 and Erk inresponse to F(ab)2IgM alone over time. FIG. 23 shows an F(ab)2 timecourse of CD20+/CD5+ population, without H₂O₂.

FIGS. 15 and 16 show different levels of BLNK phosphorylation inresponse to an external stimulus in cells with different levels ofZAP70.

FIGS. 17-21 show the kinetics of phosphorylation of PLCγ₂, S6 and Cblinthe CD20+/CD5+ cell population of CLL samples in response to B cellreceptor crosslinking with and without peroxide. FIG. 24 shows thekinetics of H₂O₂ treatment in CD20+/CD5+ population of CLL samples. FIG.25 shows the kinetics of H₂O₂ treatment in CD20+/CD5+ population of CLLsamples. These results show that F(ab)₂IgM, alone mediates an increasein the phosphorylation of rpS6, in contrast to previously evaluatedsignaling molecules. H₂O₂ alone or in combination with F(ab)₂IgMattenuates this phosphorylation. PLCγ₂ phosphorylation increased inresponse to H₂O₂ alone or to the combination with F(ab)₂IgM. Cbl has aminimal response to all treatments. Without being limited to any theory,these results suggest that a separate pathway emanates from the BCR thatregulates prpS6 and is distinct from the pathway(s) that regulate Erk,Syk/Zap, BLNK and PLCγ₂ with a distinct negative feedback loop

In summary, these results show that for CLL patient samples PMA'sactivation of p-Erk is comparable in all patients regardless of ZAP70expression and F(ab)₂IgM alone did not activate signaling. In two out ofsix CLL specimens two subpopulations of cells with distinct signalingprofiles were observed. Finally, the kinetics of activation is differentfor Syk/ZAP70, BLNK and Erk.

Example 2 CLL Patients Display Distinguishable Patterns

Isolation, storage, thawing, and equilibration of primary cells. PBMCwere isolated using density gradient separation (Ficoll-Paque Plus;Amersham Biosciences). In some embodiments, PBMC were pelleted bylow-speed centrifugation, resuspended in medium composed of 90% FCS(HyClone)+10% DMSO (Sigma-Aldrich), frozen slowly in the vapor phase ofliquid nitrogen in multiple cryotubes, and stored in liquid nitrogen.For signaling analysis of frozen samples, an individual cryotube wasthawed into 5 ml of RMPI+1% serum, counted, pelleted, and resuspended at3.3×10⁶ cells/ml. Thawed PBMC were allowed to rest at 37° C. in a CO₂incubator for 2 h before stimulation.

Modulation

At least half an hour before stimulation, 300 μA of medium containing1×10⁶ PBMC was aliquoted into flow cytometry tubes (Falcon 2052; BDBiosciences) and allowed to rest at 37° C. in a CO₂ incubator.Cross-linking of B cell receptors was achieved using goat polyclonalanti-IgM and anti-IgG F(ab′)₂ (BioSource International). When used, H₂O₂was at 3.3 mM final concentration and was added as 2 μl of a 500-mMstock solution immediately after BCR cross-linking During signaling,cells were kept in a 37° C. CO₂ incubator to allow signal transductionand phosphorylation. Signaling was stopped after 10 min. by fixing thecells. To determine basal levels of phosphorylation, unstimulated cellswere maintained in parallel with stimulated cells and fixed at timezero. For fixation, paraformaldehyde (Electron Microscopy Services) wasadded to each tube of cells to a final concentration of 1.4%. Cells werefixed for 5 min at room temperature, pelleted, permeabilized byresuspension in 2 ml of methanol for 10 min, and stored at 4° C. untilbeing stained for flow cytometry.

Flow Cytometry

Paraformaldehyde-fixed, methanol-permeabilized cells were rehydrated byaddition of 2 ml of PBS, gentle resuspension, and then centrifugation.The cells were washed and stained as in Example I except phosphospecificalexa (Ax) dye Ax488 and Ax647 (Molecular Probes) or R-PE-conjugated Abswere also used. Staining cocktails comprised fluor conjugated antibodiesspecific for p-ERK1/2(T202/Y204), and p-Syk(Y352)/Zap70(Y319),p-Lck/p-Lyn, p-BLNK, p-PLCy₂ (PLCr2) or p-S6. Detection, selection andgating of cell subsets was as described in Example I. Heat maps weregenerated using MeV (MultiExperiment Viewer) software (FIGS. 26, 27 &28).

Results

The data shown in FIGS. 26, 27 and 28 demonstrate that B-cells fromvarious CLL patients display distinguishable patterns of activatableelements as visualized by a heat map. Modulators of phosphorylationfurther define additional patterns of activatable elements that allowidentification, classification and grouping of cryptic or aberranthematopoetic populations. In FIGS. 26, 27 and 28 patient samples areindicated at the top of the heat map. Each column represents a singlepatient. CLL indicates that the sample was obtained from a patientdiagnosed with CLL. CON indicates that the sample was obtained from acontrol patient. The heat map legend is indicated at the top of thefigure and uses a shaded scale based on the log10-fold increase, ordecrease, in mean fluorescence intensity (MFI), relative to theunstimulated control (0 min). Labels to the right of the histogramindicate the phospho-protein stained and the modulator treatment forthat row. “US” indicates unstimulated. FIG. 28 illustrates theidentification of several patient clustering groups comprised of similaror distinct levels of p-PLCγ₂, p-SyK/Zap-70, p-BLNK and p-Lck. Somepatient clusterings become apparant upon modulation as illustrated bythe boxed regions.

In FIG. 28, treatment with H₂O₂ reveals a patient clustering defined bythe levels of p-PLCγ₂, p-SyK/Zap-70, p-BLNK and p-Lck (bottom rightboxed area) that are similar to those of the four control patients(bottom center box). Treatment with H₂O₂ further reveals a patientclustering that is distinct from the controls (9 patients to the left ofbottom boxed area). Modulation with H₂O₂ and BCR crosslinking definesanother patient clustering comprised of levels of p-BLNK, p-Syk andp-PLCγ₂ (top left boxed area) that are similar to the control patients(top center box) and an abberant population of responders (10 patientsto the right of top boxed area).

Example 3 Evaluation of Apoptosis Pathways in CLL Patient Samples

Current therapeutic approaches for CLL involve fludarabine-basedregimens combined with monoclonal antibodies such as rituximab.Fludarabine, a purine analog, inhibits DNA synthesis by interfering withribonucleotide reductase and DNA polymerase. Rituximab is a chimericCD20 specific antibody and has mechanistically been shown to bindcomplement, induce antibody-dependent cellular cytotoxicity (ADCC) and,in some situations, rituximab binding to CD20 inhibits proliferation andinduces cellular apoptosis (for a discussion of apoptosis see U.S. Ser.No. 61/085,789).

Cellular apoptosis in response to therapeutic agents, including but notlimited to, DNA damaging agents such as Fludarabine or biological agentssuch as Rituximab, can be measured by multiparameter flow cytometryusing fluorophore-conjugated antibodies that recognize intracellularprotein components or nodes of the apoptotic machinery. Such nodes mayinclude, but are not limited to, Caspase 3, Caspase 8, Cytochrome C,Poly ADP ribose polymerase (PARP), Bcl-2, Bcl-X, p-Chk2, p-BAD. Furtherinformation may be gathered by treating cells with a pan-caspaseinhibitor Benzyloxycarbonyl-Val-Ala-Asp (OMe) fluoromethylketone (Z-VAD.FMK) in order to reveal caspase-dependent and/or independent pathways.The profile of how the apoptotic proteins respond to treatment with atherapeutic agent can be used to inform clinical decisions.

Experimental Procedure for Measuring Response to Apoptosis of SamplesTreated with Fludarabine and Rituximab.

Cells are thawed at 37° C. in water bath until partially thawed (−50%)and are then added to RPMI/1% FBS at room temperature. Cells arecentrifuged at 900 RPM for 10 minutes, the supernatants are decanted andpellets are resuspended in 25 milliliters of media. This step isrepeated. An aliquot of cells is counted with a hemocytometer usingtrypan blue staining Cells are resuspended in RPMI/1% or 10%/FBS at thedesired concentration. After an incubation at 37° C. for 2 hr., cellsare aliquoted at a concentration of 1×10⁶ cells per well of a 96-wellplate. Cells are incubated with Fludarabine (at concentrations of 0-50μM), Rituximab (at concentrations from 0-500 μM) and/or Staurosporine,alone or in combinations, in the absence or presence of ZVAD for varioustimes. Post-incubation with drug, cells are processed for staining withcocktails of fluorochrome conjugated antibodies including CD3, CD5,CD19, CD20, CD3, CD5, CD19, CD20 (extracellular markers) andfluorochrome conjugated antibodies to the nodes/markers of apoptosisdescribed above. Cells are analyzed by flow cytometry. The details ofcell processing and flow cytometry analysis are given in U.S. Ser. No.61/085,789.

While preferred embodiments of the present invention have been shown anddescribed herein, it will be obvious to those skilled in the art thatsuch embodiments are provided by way of example only. Numerousvariations, changes, and substitutions will now occur to those skilledin the art without departing from the invention. It should be understoodthat various alternatives to the embodiments of the invention describedherein may be employed in practicing the invention. It is intended thatthe following claims define the scope of the invention and that methodsand structures within the scope of these claims and their equivalents becovered thereby.

1-50. (canceled)
 51. A method of determining a status of a primarychronic lymphoid leukemia (CLL) cell to analyze the signaling pathwaystherein comprising: contacting said CLL cell with a B-cell receptormodulator which is selected from the group consisting of Rituxan, across-linker of the B-cell receptor complex, a B-cell co-receptorcomplex, F(ab)2 IgM, IgG, IgD, polyclonal BCR antibodies, monoclonal BCRantibodies, Fc receptor derived binding elements and/or combinationsthereof; determining an activation level of an activatable element in aB-cell receptor signaling pathway in said CLL cell on a single cellbasis, said activatable element comprising p-Erk 1/2; and determiningthe status of the B-cell receptor signaling pathway in said CLL cellfrom said activation level of p-Erk 1/2.
 52. The method of claim 51,wherein the B-cell receptor modulator is F(ab)2 IgM.
 53. The method ofclaim 51, wherein the activation level of said activatable element iscompared to a normal cell contacted with said modulator.
 54. The methodof claim 51, further comprising contacting said cell with a kinase orphosphatase inhibitor.
 55. The method of claim 51, further comprisingdetermining an activation level of a plurality of activatable elements.56. The method of claim 54, wherein said kinase or phosphatase inhibitoris selected from the group consisting of adaphostin, aloisine A,alsterpaullone, aminogenistein, API-2, apigenin, arctigenin, AY-22989,BAY 61-3606, bisindolylmaleimide IX, chelerythrine,10-[4′-(N,N-Diethylamino)butyl]-2-chlorophenoxazine hydrochloride,dasatinib, 2-Dimethylamino-4,5,6,7-tetrabromo-1H-benzimidazole,5,7-Dimethoxy-3-(4-pyridinyl)quinoline dihydrochloride, edelfosine,ellagic acid, enzastaurin, ER 27319 maleate, erlotinib, ET18OCH3,fasudil, flavopiridol, gefitinib, GW 5074, H-7, H-8, H-89, HA-100,HA-1004, HA-1077, HA-1100, hydroxyfasudil, indirubin-3′-oxime,5-Iodotubercidin, kenpaullone, KN-62, KY12420, LFM-A13, lavendustin A,luteolin, LY-294002, LY294002, mallotoxin, ML-9, NSC-154020, NSC-226080,NSC-231634, NSC-664704, NSC-680410, NU6102, olomoucine, oxindole I,PD-153035, PD-98059, PD-169316, phloretin, phloridzin, piceatannol,picropodophyllin, PK1, PP1, PP2, purvalanol A, quercetin, R406, R788,rapamune, rapamycin, Ro 31-8220, roscovitine, rottlerin, SB202190,SB203580, sirolimus, sorafenib, SL327, SP600125, staurosporine, STI-571,SU-11274, SU4312, SU6656, 4,5,6,7-Tetrabromotriazole, TG101348,Triciribine, Tyrphostin AG 490, Tyrphostin AG 825, Tyrphostin AG 957,Tyrphostin AG 1024, Tyrphostin SU1498, U0126, VX-509, VX-667, VX-680,W-7, wortmannin, XL-019, XL-147, XL-184, XL-228, XL-281, XL-518, XL-647,XL-765, XL-820, XL-844, XL-880, Y-27632, ZD-1839, ZM-252868, ZM-447439,H₂O₂, siRNA, miRNA, Cantharidin, (−)-p-Bromotetramisole, Microcystin LR,Sodium Orthovanadate, Sodium Pervanadate, Vanadyl sulfate, Sodiumoxodiperoxo(1,10-phenanthroline)vanadate, bis(maltolato)oxovanadium(IV),Sodium Molybdate, Sodium Permolybdate, Sodium Tartrate, Imidazole,Sodium Fluoride, β-Glycerophosphate, Sodium Pyrophosphate Decahydrate,Calyculin A, Discodermia calyx, bpV(phen), mpV(pic), DMHV, Cypermethrin,Dephostatin, Okadaic Acid, NIPP-1,N-(9,10-Dioxo-9,10-dihydro-phenanthren-2-yl)-2,2-dimethyl-propionamide,α-Bromo-4-hydroxyacetophenone, 4-Hydroxyphenacyl Br,α-Bromo-4-methoxyacetophenone, 4-Methoxyphenacyl Br,α-Bromo-4-(carboxymethoxy)acetophenone, 4-(Carboxymethoxy)phenacyl Br,bis(4-Trifluoromethylsulfonamidophenyl)-1,4-diisopropylbenzene,phenyarsine oxide, Pyrrolidine Dithiocarbamate, and aluminum fluoride.57. The method of claim 51, wherein the activation level is selectedfrom the group consisting of glycosylation level, phosphorylation level,acetylation level, methylation level, biotinylation level, glutamylationlevel, glycylation level, hydroxylation level, isomerization level,prenylation level, myristoylation level, lipoylation level,phosphopantetheinylation level, sulfation level, ISGylation level,nitrosylation level, palmitoylation level, SUMOylation level,ubiquitination level, neddylation level, citrullination level,deamidation level, disulfide bond formation level, proteolytic cleavagelevel, translocation level, changes in protein turnover, multi-proteincomplex level, oxidation level, multi-lipid complex level, andbiochemical changes in cell membrane.
 58. The method of claim 51,wherein said activatable element further comprises a protein selectedfrom the group consisting of Akt1, Akt2, Akt3, SAPK/JNK1,2,3, p38s, Syk,ZAP70, Btk, BLNK, Lck, PLCγ, PLC1γ₂, STAT1, STAT 3, STAT 4, STAT 5, STAT6, CREB, Lyn, p-S6, Cbl, NF-κB, GSK3β, CARMA/Bcl10 and Tcl-1.
 59. Themethod of claim 54, wherein the kinase or phosphatase inhibitor is H₂O₂.60. A method of determining a status of a B-cell receptor signalingpathway of a primary CLL cell, comprising: subjecting said CLL cell to afirst modulator comprising H₂O₂; subjecting the cell to at least asecond modulator comprising F(ab)2 IgM; determining an activation levelof a phosphorylated protein, on a single cell basis, that participatesin a B-cell receptor signaling pathway in said cell, said phosphorylatedprotein comprising p-Erk 1/2; and determining the status of the B-cellreceptor signaling pathway in said CLL cell from said activation levelof p-Erk 1/2.
 61. A method of determining a status of a primary CLL cellto analyze signaling pathways therein comprising: contacting the CLLcell with a modulator which is a B-cell receptor crosslinker selectedfrom the group consisting of F(ab)2 IgM, IgG, IgD, polyclonal BCRantibodies, or monoclonal BCR antibodies, and Fc receptor derivedbinding elements; determining an activation level of an activatableelement, on a single cell basis, that participates in a tonic signalingpathway and a B-cell receptor signaling pathway in said CLL cell, saidactivatable element comprising p-Erk 1/2; and determining the status ofthe B-cell receptor signaling pathway in said CLL cell from saidactivation level of p-Erk 1/2.
 62. The method of claim 61, wherein theB-cell receptor crosslinker comprises F(ab)2 IgM.
 63. The method ofclaim 61, further comprising providing a kinase or phosphataseinhibitor.
 64. The method of claim 63, wherein the kinase or phosphataseinhibitor comprises H₂O₂.